Methods and compositions for regenerating tissues

ABSTRACT

Provided are methods for promoting the healing of injuries to tendons and ligaments by administering a NELL1 protein or a nucleic acid encoding a NELL1 protein to a subject in need thereof. Also provided are NELL1 compositions and methods for promoting tissue regeneration, promoting the healing of wounds, and enhancing fibroblast migration, proliferation, or both migration and proliferation.

CROSS REFERENCED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/380,920, filed Aug. 29, 2016, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This application generally relates to the healing of injuries, includingthose involving tendons and/or ligaments, with a regenerative protein ora nucleic acid encoding the same.

BACKGROUND OF THE INVENTION

The healing of an injury involves a multistep process whereby injuredtissue is repaired, specialized tissue is regenerated, and new tissue isreorganized into a functional unit. Wound healing is generally dividedinto the inflammatory phase, the proliferative phase, and the maturationand remodeling phase. An impairment in any one of these phases can leadto complications. Particular tissues can be more difficult to healproperly due in part to their cellular makeup, limited vasculature,and/or location in highly mobile parts of the body. Injuries to tendonsand ligaments, for example, can be particularly challenging to properlyheal.

Tendons are soft connective tissues that connect muscle to bone orcartilage, while ligaments connect bone to bone to hold the skeleton andstabilize the joints. They are composed primarily of packed collagenfibers that impart different mechanical properties based on the shapeand how they are organized. There is very little cellularity in thesetissues—mostly fibroblasts secreting the extracellular material (ECM).Ligaments have a lower collagen content (˜75%) with a random pattern,while tendons have more collagen (80-90%) and are highly organized. TheECM of both tissues are composed of Types I and III collagen, withligaments having a lesser proportion of Type I (90%) and more of typeIII (10%) in comparison to tendons which have 95-99% Type 1 and 1-5%Type III. Another key difference between these two types of connectivetissue is the presence of elastin in ligaments, which is very minimal intendons. (Lui (2015) Stem Cells and Cloning: Advances and Applications8:163-174).

Considerable basic studies have been devoted to the understanding of thedevelopment, structure, and function of these important tissues, becausetraumatic tendon/ligament injuries are extremely difficult to healcompletely. These injuries often result in permanent disability andchronic pain in human patients, while in veterinary animals such ashorses or dogs, damage to key tendon and ligaments can adversely affectthe quality of life so severely that euthanasia is necessary. Treatingtendon/ligament injuries is clinically challenging in both human andveterinary medicine because of the innate poor tissue healing response,likely due to the primary characteristics of these soft tissuesincluding: a) a low proportion of resident progenitor and stem cells; b)limited vasculature or blood vessel supply; and c) a tendency forpersistent inflammation because these are located in highly mobile bodyparts. The constant motion of the injured sites can hinder healing. Evenin cases where healing occurs, a fibrovascular scar is formed which doesnot possess the biomechanical and biochemical properties ofnormal/original tendon and/or ligament, thereby resulting in significantloss of function for the individual and increased re-injury rates. (Lui(2015); Ross (2014) Stem Cell Discovery 4:44-53)

The three most common tendon injuries in man are tearing of the rotatorcuff, injuries to the hand flexor tendon, and injuries to the Achillestendon. These are often treated by surgical repair, physicaltherapy/rehabilitation and cryotherapy. In 2004, it was estimated that45% of 32 million musculoskeletal injuries involved tendons andligaments, and that the incidence was rapidly rising due to increasedsports activities and an aging population. (September et al. (2007) Br JSports Med 41:241-246)

The most common tendon and/or ligament injuries in racehorses andperformance horses affect the suspensory ligament running behind thecannon bone, and the superficial and deep digital flexor tendons (SDFTand DDFT) running behind the back of the knee (or hock) all the way downto the navicular bone in each foot, acting as a sling for the fetlock tobear weight. (Briggs (2011) The Horse Jun. 3, 2011, pp. 1-7). Injuriescan be inflammation, sprains, strains, disruptions or tears andlacerations. Tendon/ligament injuries are estimated to be ˜46% of allsport horse injuries, and 90% of tendon/ligament injuries are to theSDFT. SDFT damage is confirmed by a veterinarian with a physical examfor lameness and ultrasound. A horse can manifest lameness, heat,sensitivity to touch, tendon swelling or thickening, or a bowed orconvex profile. Lameness correlates the degree of inflammation. (Tan(2016) The Horse Mar. 30, 2016, pp. 1-7). DDFT injuries are common inthe hoof capsule and tendon sheath and are classified as tendonenlargements, changes in shape, focal core lesions, mineralization andmarginal tears. Tears are best detected with ultrasound, MRI ortenoscopy.

Ligament injuries in horses are often proximal suspensory desmitis (PSD)in the limbs that result in acute lameness. Hind limb PSD occursfrequently in high-level dressage horses and is not very responsive toconservative therapy which consists of repeated bandaging,administration of anti-inflammatories and analgesics (only 14% returnwithout lameness for less than one year).

The repair or regeneration of tendon/ligament injuries have been aconsistent target of the increasing number of regenerative medicinetechnologies, such as stem cells, platelet rich plasma (PRP), bonemarrow aspirate concentrate, growth factors, and bioengineeredscaffolds. Techniques such as extracorporeal shock wave therapy, lowlevel laser therapy and mechanical stimulation have also been employed.(Thomas (2005) The Chronicle of the Horse 134-137; Fortier and Smith(2008) Vet Clin Equine 24:191-201; Briggs (2011); Yang et al. (2013)Birth Defects Res C Embryo Today 99(3):203-222; Lui (2015); Tan (2016);Basetto et al. (2011) Biomaterials Science and Engineering, Ch. 18, Ed.R. Pignatello, InTech, 2011, pp. 369-386) More effective treatments torestore or regenerate tendon/ligament tissues after injury are needed.

SUMMARY OF THE INVENTION

Compositions comprising a variant NELL1 peptide or a nucleic acidencoding the same are provided. The variant NELL1 peptide lacks at leastone of the carboxy-terminal von Willebrand factor, type C (VWC) domainsof a NELL1 protein. In some embodiments of the invention, the variantNELL1 peptide lacks both carboxy-terminal VWC domains. In some of theseembodiments, the variant NELL1 peptide lacks the carboxy-terminal 179amino acid residues. In particular aspects of the invention, the variantNELL1 peptide has at least 75% sequence identity to the disclosed SEQ IDNO: 17 or 18 and one of the following properties: enhanced efficacy intissue regeneration, promotion of wound healing, easier purification,higher yield, and less aggregate formation, when compared to afull-length NELL1 protein. In some of these embodiments, the variantNELL1 peptide comprises or consists essentially of SEQ ID NO: 17 or 18.Also provided herein are pharmaceutical compositions and kits comprisingthe variant NELL1 peptide or a nucleic acid encoding the same.

The variant NELL1 peptides or nucleic acids encoding the same find usein regenerating injured tissues, promoting the maturation of aprogenitor cell, enhancing the migration and/or proliferation offibroblast cells, and promoting the healing of a wound, such as aninjury to a tendon or a ligament. In particular aspects of theinvention, the wound is an open wound. In certain embodiments, the woundcomprises an injury to skeletal muscle, cartilage, bone, skin, tendon,ligament or a combination thereof. In some aspects, the variant NELL1peptide or nucleic acid encoding the same is administered locally to thewound. In particular embodiments, the subject in need of promotion ofhealing of a wound is a mammal, such as a human or a horse. The variantNELL1 peptide or nucleic acid encoding the same can be administered tothe wound about two days after the injury.

Methods for promoting the healing of an injury to a tendon or ligamentin a subject in need thereof are provided herein. These presentlydisclosed methods involve the administration of an effective amount of aNELL1 peptide or a nucleic acid encoding a NELL1 peptide to the subject.The subject can be a mammal, such as a human or a horse. The methodsfind use in treating various injuries in humans, including injuries toAchilles tendons. Horses, especially racehorses, are particularly proneto tendon and ligament injuries, such as the superficial digital flexortendon or deep digital flexor tendon, and the presently disclosedmethods are useful in promoting the healing of these injuries. The NELL1peptide or nucleic acid encoding the same can be administered via localinjection to the area surrounding the injured tendon or ligament or canbe incorporated into a matrix, such as a wound dressing and applieddirectly to the injured tissue. The NELL1 peptide or nucleic acidencoding the same can be administered to the tendon or ligament injuryabout seven days after the injury.

Methods for enhancing the migration and/or proliferation of fibroblastcells with a NELL1 peptide or a nucleic acid encoding the same are alsoprovided herein. In particular aspects of the invention, the fibroblastcell is a dermal fibroblast or a ligament fibroblast. In someembodiments, the migration of the fibroblasts towards a wound area isenhanced with a NELL1 peptide or a nucleic acid encoding the same.

The foregoing is a summary and thus contains, by necessity,simplifications, generalizations, and omissions of detail; consequently,those skilled in the art will appreciate that the summary isillustrative and is not intended to be in any way limiting. Otheraspects, features, and advantages of the methods, compositions and/ordevices and/or other subject matter described herein will becomeapparent in the teachings set forth herein. The summary is provided tointroduce a selection of concepts in a simplified form that are furtherdescribed below in the Detailed Description. This summary is notintended to identify key features or essential features of the claimedsubject matter, nor is it intended to be used as an aid in determiningthe scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the general structure of the human nel-like 1 isoform 1(SEQ ID NO: 2).

FIGS. 2A-2D depict purified recombinant horse NELL1 protein aa3-791(full-length; FIGS. 2A and 2B) and horse NEL1 variant protein aa3-612(FIGS. 2C and 2D). FIGS. 2A and 2C depict sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), whereas FIGS. 2B and 2Ddepict Western Blots using mouse anti-GST antibody (1 μg/ml) to the GSTtag attached to the NELL1 protein, followed by Jackson anti-mouseIgG-HRP (1500×). The lanes in FIG. 2A are as follows: M=molecularmarkers (kilodaltons or kDa) 10 μl/lane; lane 1=NELL1 protein aa3-791 (5μl/lane); and lanes 2-5 are loading control proteins at 0.1 μg/lane, 0.2μg/lane, 0.5 μg/lane, and 1.0 μg/lane, respectively. The lanes in FIG.2B are as follows: M=molecular markers (kDa); lane 1 is NELL1 aa3-791protein (5 μl); and lane 2 is GST protein. The lanes in FIG. 2C are asfollows: M=molecular markers 10 μl/lane (kDa); lane 1=NELL1 proteinaa3-612 (5 μl/lane); and lanes 2-5 are loading control proteins at 0.1μg/lane, 0.2 μg/lane, 0.5 μg/lane, and 1.0 μg/lane, respectively. Thelanes in FIG. 2D are as follows: M=molecular markers (kDa); lane 1 isNELL1 aa3-612 protein (5 μl); and lane 2 is GST protein.

FIGS. 3A-3D depict effects of full-length NELL1 protein (aa3-791) or aNELL1 protein variant (aa3-612) on wound healing assays using diabeticdermal fibroblasts (FIGS. 3A and 3B), normal dermal fibroblasts (FIG.3C), and normal ligament fibroblasts (FIG. 3D). FIG. 3A demonstrates adose-dependent increase in wound healing on Type 1 diabetic human dermalfibroblasts in an in vitro wound scratch assay with Nell1 variantprotein (aa3-612). There is increased migration of the diabetic dermalfibroblasts into the wound area when treated with 100 ng/ml (line b),200 ng/ml (line c), and 300 ng/ml (line d) of the protein, compared tothe untreated controls (line a). FIGS. 3B, 3C, and 3D demonstrates anincreased wound healing effect on Type 1 diabetic human dermalfibroblasts, normal human dermal fibroblasts, and normal human ligamentfibroblasts, respectively, in an in vitro wound scratch assay by 300ng/ml of NELL1 variant protein (aa3-612; line C), compared to thefull-length NELL1 protein at the same dose (line B) and the untreatedcontrol (line A).

FIG. 4 provides an illustration of the wound grid on each side of thehorses indicating the three 4-cm diameter (A, B, C or E, F, G) and the2-cm diameter cutaneous wounds (W, Y, W and X, or Y and Z) andtreatments with control, full-length NELL1 (NFL), or NELL1 variant(NV1). Top grids were for horses 1-4 and the bottom grids were forhorses 5-8.

FIG. 5 shows horse body wounds at day 42 post-treatment showing improvedhealing with NELL1 variant (NV1) compared to full-length NELL1 (NFL) andcontrols.

FIG. 6 shows a graph of the natural log values of the inner area inhorse wounds that remain unhealed on days post-treatment withfull-length (lines 3 and 4) or variant NELL1 protein (lines 5 and 6) anduntreated control wounds (lines 1 and 2).

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

While the present invention may be embodied in many different forms,disclosed herein are specific illustrative embodiments thereof thatexemplify the principles of the invention. It should be emphasized thatthe present invention is not limited to the specific embodimentsillustrated. Moreover, any section headings used herein are fororganizational purposes and are not to be construed as limiting thesubject matter described. Finally, for the purposes of the instantdisclosure all identifying sequence Accession numbers may be found inthe NCBI Reference Sequence (RefSeq) database and/or the NCBI GenBankarchival sequence database unless otherwise noted.

II. NELL1

The neural epidermal growth-factor-like (nel) gene was first detected inneural tissue from an embryonic chicken cDNA library, and its humanortholog neural epidermal growth-factor-like 1 (NEL-like 1, NELL1) wasdiscovered later in B-cells. Studies have reported the presence of NELL1in various fetal and adult organs, including, but not limited to,skeletal and cardiac muscle, skin, the brain, kidneys, colon, thymus,lung, and small intestine.

The human NELL1 gene encodes an 810-amino acid polypeptide. Generally,the arrangement of the functional domains of the NELL1 protein bearsresemblance to thrombospondin-1 (THBS1) and consists of a thrombospondinN-terminal domain (TSPN) and several von Willebrand factor, type C(VWC), and epidermal growth-factor (EGF) domains. A domain is a regionof a protein with a characteristic primary structure and function.

Additional studies have shown that there are at least two human NELL1transcript variants encoding different isoforms. In humans, the nel-like1 isoform 1 precursor transcript variant (set forth in SEQ ID NO: 1)represents the longer transcript (set forth in GenBank Acc. No.NM_006157) and encodes the longer isoform 1 (set forth in SEQ ID NO: 2).

FIG. 1 shows the general structure of human nel-like 1 isoform 1 (SEQ IDNO: 2). The conserved domains reside in seven regions of the isoform 1peptide and include: (1) a TSPN domain/Laminin G superfamily domain; (2)a VWC domain; (3) four EGF-like domains; and (4) a VWC domain. NELL1also comprises a secretion signal peptide domain (amino acid residues1-16 of SEQ ID NO: 2) that is generally involved in transport of theprotein to cell organelles where it is processed for secretion outsidethe cell.

The first conserved domain region comprises amino acids (amino acids 29to 213 of SEQ ID NO: 2) that are most similar to a thrombospondinN-terminal-like domain. Thrombospondins are a family of related,adhesive glycoproteins, which are synthesized, secreted and incorporatedinto the (ECM) of a variety of cells, including alpha granules ofplatelets following thrombin activation and endothelial cells. Theyinteract with a number of blood coagulation factors and anticoagulantfactors, and are involved in cell adhesion, platelet aggregation, cellproliferation, angiogenesis, tumor metastasis, vascular smooth musclegrowth and tissue repair. The first conserved domain also comprisesamino acids (amino acids 82 to 206; amino acids 98 to 209 of SEQ ID NO:2) that are similar to a Laminin G-like domain. Laminin G-like (LamG)domains usually are Ca²⁺ mediated receptors that can have binding sitesfor steroids, β1-integrins, heparin, sulfatides, fibulin-1, andα-dystroglycans. Proteins that contain LamG domains serve a variety ofpurposes, including signal transduction via cell-surface steroidreceptors, adhesion, migration and differentiation through mediation ofcell adhesion molecules.

Studies show that NELL1 signaling involves an integrin-related moleculeand tyrosine kinases that are triggered by NELL1 binding to a NELL1specific receptor and a subsequent formation of an extracellularcomplex. As thus far understood, in human NELL1 (hNELL1), the laminin Gdomain comprises about 128 amino acid residues that show a high degreeof similarity to the laminin G domain of extracellular matrix (ECM)proteins; such as human laminin α3 chain (hLAMA3), mouse laminin α3chain (mLAMA3), human collagen 11 α3 chain (hCOLA1), and humanthrombospondin-1 (hTSP1). This complex facilitates either activation oftyrosine kinases, inactivation of tyrosine phosphatases, orintracellular recruitment of tyrosine-phosphorylated proteins. Theligand bound integrin (cell surface receptors that interact with ECMproteins such as, for example, laminin 5, fibronectin, vitronectin,TSP1/2) transduces the signals through activation of the focal adhesionkinase (FAK) followed by indirect activation of the Ras-MAPK cascade,and then leads to osteogenic differentiation through Runx2; the lamininG domain is believed to play a role in the interaction between integrinsand a 67 kDa laminin receptor (Shen et al. (2012) J Cell Biochem113:3620-3628).

The second conserved domain (amino acids 273 to 331 of SEQ ID NO: 2) andseventh conserved domain (amino acids 701 to 749 of SEQ ID NO: 2) aresimilar to von Willebrand factor type C (VWC) domains, also known aschordin-like repeats. An additional VWC domain is also found from aminoacid residues 634 to 686 of SEQ ID NO: 2. VWC domains occur in numerousproteins of diverse functions and have been associated with facilitatingprotein oligomerization.

The third conserved domain (amino acids 434 to 466 of SEQ ID NO: 2),fourth conserved domain (amino acids 478 to 512 of SEQ ID NO: 2), fifthconserved domain (amino acids 549 to 586 of SEQ ID NO: 2), and sixthconserved domain (amino acids 596 to 627 of SEQ ID NO: 2) are similar toa calcium-binding EGF-like domain. Calcium-binding EGF-like domains arepresent in a large number of membrane-bound and extracellular (mostlyanimal) proteins. Many of these proteins require calcium for theirbiological function. Calcium-binding sites have been found to be locatedat the N-terminus of particular EGF-like domains, suggestingcalcium-binding may be crucial for numerous protein-proteininteractions. Six conserved core cysteines form three disulfide bridgesas in non-calcium-binding EGF domains whose structures are very similar.The calcium-binding EGF-like domains of NELL1 bind protein kinase Cbeta, which is typically involved in cell signaling pathways in growthand differentiation.

The nel-like 1 isoform 2 precursor transcript variant (set forth inGenBank Acc. No. NM_201551 and SEQ ID NO: 3) lacks an alternate in-frameexon compared to variant 1. The resulting isoform 2 (set forth in SEQ IDNO: 4), which has the same N- and C-termini as isoform 1 but is shortercompared to isoform 1, has six conserved regions including a TSPNdomain/LamG superfamily domain (amino acids 29 to 213 of SEQ ID NO: 4);VWC domains (amino acids 273 to 331 of SEQ ID NO: 4; amino acids 654 to702 of SEQ ID NO: 4); and calcium-binding EGF-like domains (amino acids478 to 512 of SEQ ID NO: 4; amino acids 434 to 466 of SEQ ID NO: 4;amino acids 549 to 580 of SEQ ID NO: 4).

NELL1 and its orthologs are found across several species including Homosapiens (man), Bos taurus (cow; the nucleic acid sequence of which isset forth in GenBank Acc. No. XM_002699102 and the amino acid sequenceis set forth in SEQ ID NO: 19), Equus caballus (horse; the nucleic acidsequence of isoforms 1 and 2 are set forth in GenBank Acc. Nos.XM_001504986 and XM_001504987, respectively, and in SEQ ID NO: 5 and 7,respectively; the amino acid sequences are set forth in SEQ ID NO: 6 and8, respectively), Macaca mulatta (rhesus monkey; the nucleic acidsequence of isoforms 1, 2, 3, and 4 are set forth in GenBank Acc. Nos.XM_002799606, XM_001092428, XM_001092540, and XM_001092655,respectively), Mus musculus (mouse; the nucleic acid sequence of whichis set forth in GenBank Acc. No. NM_001037906 and in SEQ ID NO: 9; theamino acid sequence of which is set forth in SEQ ID NO: 10), Rattusnorvegicus (rat; the nucleic acid sequence of which is set forth inGenBank Acc. No. NM_031069 and in SEQ ID NO: 11; the amino acid sequenceof which is set forth in SEQ ID NO: 12), Pan troglodytes (chimpanzee;the nucleic acid sequence of which is set forth in GenBank Acc. No.XM_508331.2), Felis catus (cat; the amino acid sequences of isoform 1and 2 are set forth in GenBank Acc. Nos. XP_003993117.1 andXP_003993118.1, and SEQ ID NOs: 13 and 14, respectively, Canis lupusfamiliaris (dog; the amino acid sequence is set forth in GenBank Acc.No. XP_534090 and SEQ ID NO: 15), and Ovis aries (sheep; the amino acidsequence is set forth in GenBank Acc. No. XP_004019490 and SEQ ID NO:16).

NELL1 is a signaling protein that mediates tissue growth and maturationin a variety of tissues such as bone, cartilage, heart and skeletalmuscle—during fetal development and the healing of acute injuries inadult tissues (Desai et al. (2006) Hum Mol Genet 15(8):1329-1341; Siu etal. (2011) Tissue Eng Part A 17(7-8):1123-1135; Siu et al. (2012) TissueEng Part A 18(3-4):252-261; Xue et al. (2011) Bone 48(3):485-495; Li etal. (2011) Plast Reconstr Surg 127(2):580-587; Turner et al. (2013)Cells Tissues and Organs 198(4):249-265). During early development,NELL1 regulates the production of many components of the extracellularmatrix (ECM) which collectively serve as an architectural framework andcommunication highway to mediate new tissue formation.

In vitro studies on three-dimensional human skin models exposed to UVradiation demonstrated that NELL1 reduces levels of key pro-inflammatorymolecules (e.g. IL1-β, IL8) after adult tissue injury (Mitchell et al.(2012) Abstract, 70^(th) Annual Meeting of the American Academy ofDermatology, San Diego, Calif., Mar. 16-20). Human genome-wideassociation studies have also suggested that in certain geneticpopulations NELL1 plays a role in controlling severe inflammatoryconditions (Franke et al. (2007) PLoS ONE 2(8):e691).

Multiple effects of NELL1 are believed to contribute to its ability toheal injuries to tendons and/or ligaments. Such effects include itspro-angiogenic activity, which stimulates blood vessel formation inareas having poor blood supply. NELL1 accomplishes this via effects onVEGF and perivascular stem cells (Askarinam et al. (2013) Tissue Eng A19(11-12):1386-1397). The effect of NELL1 in perivascular cells isparticularly important in tendon regeneration because the capillaries oftendons contain perivascular stem cells that express tendon- andstem/precursor cell-like characteristics which could be the source ofthe formation/regeneration of new tendon tissue (Tempfer et al. (2009)Histochem Cell Biol 131(6):733-741). While not being held by any theoryor mechanism of action, it is believed that NELL1 can also stimulate theproliferation and migration of tendon precursor stem cells, fibroblastsor perivascular stem cells, contributing to the healing of tendons(Nemoto et al. (2013) J Equine Sci 24(2):17-24).

NELL1 induces the production of molecules in the extracellular matrixthat are key structural components of tendons and ligaments or moleculesthat regulate the production of components and theirassembly/organization into the correct functional architecture. Some ofthe known genes in the NELL1 pathway directly impact tendon or ligamentdevelopment, structure, function, repair and regeneration afterinjuries. This list includes tenascin C, collagen V, Bmp7, periostin orosteoblast specific factor 2, and Prg4 (lubricin). Tenascin C is aglycoprotein abundant in tissues with high tensile strength and subjectto compression stress. It is believed to be a key factor in tendonhealing due to its ability to promote fibroblast/tendon cellproliferation and migration. Tenascin C is a genetic determinant ofAchilles heel tendinopathies and ruptures and is linked to tissueresponse to mechanical loading, probably by regulating cell-ECMinteractions (September et al. (2007); Taylor et al. (2009) BUMMusculoskeletal Disorders 10(27):1-10; Juneja & Veillette (2013)Arthritis 2013:1-30; Nemoto et al. (2013)). Collagen V, specificallyCol5a1 and Col5a3, is a component of fibrillary collagen that regulatescollagen fiber assembly and diameter in tendons and ligaments (Septemberet al. (2007); Connizzo et al. (2015) J Orthopaedic Research 33:882-888;Sun et al. (2015) Am J Pathol 185:1436-1447). Bmp7 is a bonemorphogenetic protein that serves as a growth factor promoting cellgrowth and differentiation. It facilitates tendon-bone integration.Other studies show it increases collagen type 1 production and cellactivity (Pauly et al. (2012) J Shoulder Elbow Surg 21(4):464-473;Schwarting et al. (2015) PLoS One 10(2):1-17). Periostin or osteoblastspecific factor 2 is a matricellular protein that is abundant incollagen rich connective tissue, where it is essential for proper ECMsynthesis, collagen 1 fibrillogenesis, and tendon crosslinking.(Hamilton et al. (2008) J Cell Commun Signal 2:9-17; Juneja & Veilette(2013)). Prg4 (lubricin) is a proteoglycan that plays a role in boundarylubrication in articulating joints and tendon gliding. Its absence leadsto decreased lubrication, which causes tissue damage, matrix remodelingand dystrophic calcification. (Juneja & Veillete (2013)). NELL1modulates the inflammatory response via the downregulation of cytokinessuch as IL-1 beta and IL8 (Mitchell et al. (2012)). Tendon and ligamentinjuries are prone to prolonged inflammation because the injured sitesare often subjected to constant motion. NELL1 downregulates matrixmetalloproteinases (e.g. MMP1), which degrade the collagen in tendonECM. MMPs are needed to maintain ECM homeostasis, but tendon injury canlead to an imbalance or dysregulation so that high levels can furtherdegrade tendon architecture and function. (Mitchell et al. (2012); Daviset al. (2013) J Applied Physiol 115(6):884-891).

Disclosed herein is the discovery that NELL1 also enhances the migrationand/or the proliferation of fibroblast cells (e.g., ligamentfibroblasts, dermal fibroblasts from normal individuals and type 1diabetic patients). This effect can serve to promote wound healing byincreasing the numbers of fibroblasts within a wound area, thusenhancing the contraction and closure of the wound. The proliferationand migration of fibroblasts into the provisional wound matrix/fibrinclot are critical processes that are triggered early in wound healing(e.g., few days after injury in human skin) and have significant rolesin supporting other biological processes throughout normal woundhealing, including the degradation of the fibrin clot, secretion of newextracellular matrix and collagen-rich structures to support theactivities of other cells, and wound contraction. The presentlydisclosed methods and compositions utilize a NELL1 peptide or a nucleicacid molecule encoding the same to promote the healing of injuries totendons or ligaments or to enhance the migration and/or proliferation offibroblasts. Certain NELL1 variants also find broader use inregenerating tissues, promoting healing of wounds, and promoting thematuration of progenitor cells.

A peptide, polypeptide, or protein is a sequence of subunit amino acids,amino acid analogs, or peptidomimetics. A peptidomimetic is a smallprotein-like chain designed to mimic a peptide. A peptidomimetictypically arises from modification of an existing peptide in order toalter the molecule's properties.

A peptide, polypeptide or protein can also be amino acid polymers inwhich one or more amino acid residue is an artificial chemical analogueof a corresponding naturally occurring amino acid, as well as tonaturally-occurring amino acid polymers. A polypeptide, peptide orprotein is inclusive of modifications including, but not limited to,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation, phosphorylation, andADP-ribosylation. It will be appreciated, as is well known and as notedabove, that polypeptides may not be entirely linear. For instance,polypeptides may be branched as a result of ubiquitination, and they maybe circular, with or without branching, generally as a result ofpost-translational events, including natural processing events andevents brought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translation natural processes and by entirely synthetic methods,as well.

A NELL1 peptide, NELL1 polypeptide, or NELL1 protein is anaturally-occurring NELL1 protein, or a variant or fragment thereof thatretains the ability to promote the healing of injuries to tendons and/orligaments. In some embodiments, the NELL1 peptide exhibits any one ofthe activities selected from the group consisting of: stimulation of ECMproduction (e.g., through the upregulation of at least one of tenascins,proteoglycans, elastin, glycosaminoglycans, including epidermalhyaluronic acid, and collagens), reduction in the levels of inflammatorymediators (e.g., IL-1β and IL-8), reduction in the levels of matrixmetalloproteinases (e.g., MMP1), and enhancing the migration and/orproliferation of fibroblasts. In other embodiments, the NELL1 peptidecan also exhibit at least one of the activities selected from the groupconsisting of binding to PKC-beta, stimulation of differentiation of aprecursor cell (e.g., skeletal satellite cell, osteoblast precursor,perivascular stem cell, or tendon precursor stem cell) to maturity, andstimulation of angiogenesis. To determine whether a peptide exhibits anyone of these activities, any method known in the art useful formeasuring these activities can be used.

Suitable assays for determining if a given peptide can stimulate ECMproduction and reduce the levels of inflammatory mediators or MMPsinclude assays that measure transcript levels (e.g., quantitativepolymerase chain reaction) or levels of the protein (e.g., enzyme-linkedimmunoassay) directly or indirectly (by measuring the activity of theprotein), including those that are described elsewhere herein.

Suitable assays for assessing the binding of NELL1 to PKC beta isdescribed in e.g., Kuroda et al. (1999) Biochem Biophys Res Comm265:752-757. For example, protein-protein interactions can be analyzedby using the yeast two-hybrid system. Briefly, a NELL1 protein can befused with GAL4 activating domain and the regulatory domain of PKC canbe fused with the GAL4 DNA-binding domain.

In other embodiments, the NELL1 peptide stimulates the differentiationof precursor cells, such as skeletal satellite cells, osteoblastprecursors, perivascular stem cells, and tendon precursor stem cells, tomaturity. The maturity of cells can be assessed cellularly (histology)and molecularly (expression of cell-specific proteins or extracellularmatrix materials).

Suitable assays for determining if a peptide is capable of promoting thehealing of tendon and/or ligament injuries include those known in theart (Nemoto et al. (2013); Taylor et al. (2009); Yanming et al. (2007)Nature Medicine 13:1219-1227; Tempfer et al. (2009)) and disclosedelsewhere herein.

Suitable assays for determining if a NELL1 peptide can promote themigration and/or proliferation of fibroblasts include wound scratchassays known in the art and described elsewhere herein.

The NELL1 peptide may be a naturally-occurring (i.e., wild-type) NELL1protein or an active variant or fragment thereof. Naturally refers to asfound in nature; wild-type; innately or inherently. Anaturally-occurring NELL1 peptide may be purified from a natural sourceor may be a peptide that has been recombinantly or syntheticallyproduced that has the same amino acid sequence as a NELL1 peptide foundin nature.

A polynucleotide can be a singular nucleic acid, as well as pluralnucleic acids, and refers to a nucleic acid molecule or construct, e.g.,messenger RNA (mRNA), complementary DNA (cDNA), plasmid DNA (pDNA), orshort interfering RNA (siRNA). A polynucleotide can be single-strandedor double-stranded, linear or circular and can be comprised of DNA, RNA,or a combination thereof. A polynucleotide can comprise a conventionalphosphodiester bond or a non-conventional bond (e.g., an amide bond,such as found in peptide nucleic acids (PNA)). A nucleic acid can be anyone or more nucleic acid segments, e.g., DNA or RNA fragments, presentin a polynucleotide. The polynucleotide can contain modified nucleicacids, such as phosphorothioate, phosphate, ring atom modifiedderivatives, and the like. The polynucleotide can be a naturallyoccurring polynucleotide (i.e., one existing in nature without humanintervention), a recombinant polynucleotide (i.e., one existing withhuman intervention), or a synthetically derived polynucleotide.

An isolated material can refer to a nucleic acid, peptide, polypeptide,or protein, which is: (1) substantially or essentially free fromcomponents that normally accompany or interact with it as found in itsnaturally occurring environment. Substantially free or essentially freerefer to considerably or significantly free of, or more than about 95%free of, or more than about 99% free of. The isolated materialoptionally comprises material not found with the material in its naturalenvironment; or (2) if the material is in its natural environment, thematerial has been synthetically (non-naturally) altered by deliberatehuman intervention to a composition and/or placed at a location in thecell (e.g., genome or subcellular organelle) not native to a materialfound in that environment. The alteration to yield the syntheticmaterial may be performed on the material within, or removed, from itsnatural state. For example, a naturally occurring nucleic acid becomesan isolated nucleic acid if it is altered, or if it is transcribed fromDNA that has been altered, by means of human intervention performedwithin the cell from which it originates. See, for example, Compoundsand Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec,U.S. Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting inEukaryotic Cells; Zarling et al., PCT/US93/03868, each of which isincorporated by reference herein. Likewise, a naturally occurringnucleic acid (for example, a promoter) becomes isolated if it isintroduced by non-naturally occurring means to a locus of the genome notnative to that nucleic acid.

Fragments and variants of native (i.e., naturally-occurring) NELLpolypeptides can be employed in the various methods and compositions ofthe invention. A fragment is intended a portion of a polynucleotide or aportion of a polypeptide. Fragments of a polynucleotide may encodepolypeptide fragments that retain the biological activity of the nativepolypeptide. A fragment of a polynucleotide that encodes a biologicallyactive portion of a NELL1 polypeptide will encode at least 15, 25, 30,50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, or 800 contiguous amino acids, or up to the total number of aminoacids present in a full-length NELL1 polypeptide. In certainembodiments, the NELL1 fragment is 610 amino acids in length.

A fragment of a native NELL1 polypeptide can be prepared by isolating aportion of a polynucleotide encoding the portion of the NELL1polypeptide and expressing the encoded portion of the polypeptide (e.g.,by recombinant expression in vitro). Polynucleotides that encodefragments of a NELL1 polypeptide can comprise nucleotide sequencescomprising at least 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, or 2400 contiguousnucleotides, or up to the number of nucleotides present in a full-lengthNELL1 nucleotide sequence. In some embodiments, the fragment lacks thefirst amino acid residue, or the first 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, or 45 amino acidresidues from the amino terminal end of the NELL1 protein. In someembodiments, the fragment lacks the last 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125,130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200, 205, 210, 220, 230, 240, 250, 260 or more amino acid residues. Incertain embodiments, the fragment of a NELL1 protein lacks the mostcarboxy-terminal 179 amino acid residues from the end of the protein. Inother embodiments, the NELL1 protein fragment lacks the first two aminoacid residues from the amino terminal end and the last 179 amino acidresidues from the carboxy terminal end of the protein. In someembodiments, the NELL1 protein fragment has 610 amino acid residues.

The inventors determined that removal of 179 amino acid residues fromthe carboxy-terminus of the Equus caballus NELL1 isoform 1 proteinunexpectedly provided a higher yield and easier purification duringmanufacture of the protein. Without being bound by any theory ormechanism of action, it is believed that the removal of thecarboxy-terminal domains led to decreased formation of aggregates of theprotein. Although NELL1 protein naturally oligomerizes into trimers,which are functional, aggregates of NELL1 protein refer to large,higher-ordered macromolecular complexes that prevent or reduce thefunction of the protein or make the protein products difficult toextract and purify. The NELL1 protein lacking the C-terminal 179 aminoacid residues is also unexpectedly more efficacious than full-lengthNELL1 protein in horse body wound healing studies and fibroblast woundscratch assays. Thus, in specific embodiments, the NELL1 proteinfragment lacks the last 179 amino acid residues from the carboxyterminus. In some of these embodiments, the NELL1 protein fragment alsolacks the first two amino acid residues from the amino terminus. Thesequence of this horse NELL1 fragment is set forth in SEQ ID NO: 18. Inother embodiments, the NELL1 protein fragment lacks the first 21 aminoacid residues from the amino terminus and the last 179 amino acidresidues from the carboxy terminus. The sequence of this human NELL1fragment is set forth in SEQ ID NO: 17. In certain embodiments, theNELL1 protein fragment lacks at least one of the two carboxy-terminalVWC domains (located at amino acid residues 634-686 and 701-749 of SEQID NO: 2). In some of these embodiments, the NELL1 protein fragmentlacks both of these carboxy-terminal VWC domains. Compositionscomprising these NELL1 fragments are contemplated herein and methods forusing these NELL1 fragments for regenerating tissues (e.g., bone,cartilage, heart, vasculature, skeletal muscle), promoting thematuration of progenitor cells for various tissues (e.g., bone,cartilage, heart, vasculature, skeletal muscle, tendons, ligaments), andpromoting the migration and/or proliferation of fibroblasts are alsocontemplated herein.

In those embodiments wherein a variant NELL1 protein lacks at least oneC-terminal VWC domain, the variant NELL1 protein exhibits at least oneof the following characteristics: enhanced efficacy in tissueregeneration and/or promotion of wound healing, easier purification,higher yield, less aggregate formation, and enhanced efficacy infibroblast migration and/or proliferation, when compared to afull-length NELL1 protein. An easier purification includes apurification process whereby a single polypeptide species issubstantially separated from other polypeptide species or a natural orsynthetic milieu comprising the single polypeptide species and otherpolypeptide species that comprises fewer steps required for substantialseparation or wherein the time required for at least one of the steps inthe separation is reduced. An easier purification also refers to apurification process which results in a higher yield of thesubstantially purified or separated polypeptide species. The terms“substantially purified” or “substantially separated” when used inreference to a single polypeptide species refers to a level ofpurification whereby the single polypeptide species represents at leastabout 70% of a total population of polypeptide species within a sample,including but not limited to at least about 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greaterof a total population of polypeptide species within a sample. A yield ofa protein product from a purification process refers to the overallconcentration of the polypeptide within a solution. The higher theconcentration of the polypeptide within the solution, the more yield isobtained. If a polypeptide is present within a solution at <0.1 μg/μl,the protein is considered difficult to produce and purify. Thus, in someembodiments, a variant NELL1 protein that lacks at least one C-terminalVWC domain exhibits the ability to be purified using conventionalpurification means known in the art, such as those methods describedelsewhere herein, to a concentration greater than 0.1 μg/μl. In some ofthese embodiments, a variant NELL1 protein has the ability to bepurified using conventional purification means known in the art to aconcentration of about 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18,0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30μg/μl, or greater. In certain embodiments, a variant NELL1 proteinlacking at least one C-terminal VWC domain exhibits both a higher yieldand a greater purity as compared to a full-length NELL1 proteinfollowing a purification process.

Variant sequences have a high degree of sequence similarity. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of a NELL1 polypeptide. Variants such as these can beidentified with the use of well-known molecular biology techniques, suchas, for example, polymerase chain reaction (PCR) and hybridizationtechniques. Variant polynucleotides also include synthetically derivednucleotide sequences, such as those generated, for example, by usingsite-directed mutagenesis. In some embodiments, the variantpolynucleotide still encodes a NELL1 polypeptide or a fragment thereof.Generally, variants of a particular polynucleotide will have at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular polynucleotide as determined by sequence alignment programsand parameters described elsewhere herein.

Variants of a particular polynucleotide can also be evaluated bycomparison of the percent sequence identity between the polypeptideencoded by a variant polynucleotide and the polypeptide encoded by thereference polynucleotide. Thus, variants include, for example,polynucleotides that encode a polypeptide with a given percent sequenceidentity to a native NELL1 polypeptide. Percent sequence identitybetween any two polypeptides can be calculated using sequence alignmentprograms and parameters described herein. Where any given pair ofpolynucleotides is evaluated by comparison of the percent sequenceidentity shared by the two polypeptides they encode, the percentsequence identity between the two encoded polypeptides is at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

A variant polypeptide is a polypeptide derived from the nativepolypeptide by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativepolypeptide; deletion or addition of one or more amino acids at one ormore sites in the native polypeptide; or substitution of one or moreamino acids at one or more sites in the native polypeptide. The activityof variant NELL1 polypeptides can be assessed using the methodsdisclosed herein to determine if the variant is biologically active.Such variants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variants of a native NELL1polypeptide will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the amino acid sequence for the native polypeptideas determined by sequence alignment programs and parameters describedelsewhere herein. A biologically active variant of a polypeptide maydiffer from that polypeptide by as few as 1-15 amino acid residues, asfew as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1amino acid residue.

Biologically active variants of the NELL1 fragments disclosed herein(i.e., those lacking at least one of the two VWC domains at the carboxyterminus of NELL1) are also contemplated herein and may have at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the aminoacid sequence for the active NELL1 fragment (e.g., SEQ ID NO: 17 or 18).

Polypeptides may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of native NELL1 polypeptides can be prepared bymutations in the DNA. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to appropriateamino acid substitutions that do not affect biological activity of thepolypeptide of interest may be found in the model of Dayhoff et al.(1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res.Found., Washington, D.C.). Conservative substitutions, such asexchanging one amino acid with another having similar properties, may bepreferable.

Generally, the mutations made in the polynucleotide encoding the variantNELL1 polypeptide should not place the sequence out of reading frame,and/or create complementary regions that could produce secondary mRNAstructure. See, EP Patent Application Publication No. 75,444.

Variant NELL1 polynucleotides and polypeptides also encompass sequencesand polypeptides derived from a mutagenic and recombinogenic proceduresuch as DNA shuffling. With such a procedure, one or more differentNELL1 coding sequences can be manipulated to create peptides that can beevaluated to determine if it retains NELL1 activity. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Variant NELL1 polynucleotides and polypeptides also encompass sequencesand polypeptides derived from gene editing systems, such as CRISPR/Cassystem.

Sequence identity in the context of two polynucleotides or polypeptidesequences makes reference to the residues in the two sequences that arethe same when aligned for maximum correspondence over a specifiedcomparison window. When percentage of sequence identity is used inreference to polypeptides it is recognized that residue positions whichare not identical often differ by conservative amino acid substitutions,where amino acid residues are substituted for other amino acid residueswith similar chemical properties (e.g., charge or hydrophobicity) andtherefore do not change the functional properties of the molecule. Whensequences differ in conservative substitutions, the percent sequenceidentity may be adjusted upwards to correct for the conservative natureof the substitution. Sequences that differ by such conservativesubstitutions are said to have sequence similarity or similarity. Meansfor making this adjustment are well known to those of skill in the art.Typically, this involves scoring a conservative substitution as apartial rather than a full mismatch, thereby increasing the percentagesequence identity. Thus, for example, where an identical amino acid isgiven a score of 1 and a non-conservative substitution is given a scoreof zero, a conservative substitution is given a score between zeroand 1. The scoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

Percentage of sequence identity is the value determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison, and multiplyingthe result by 100 to yield the percentage of sequence identity.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3; % identity and %similarity for an amino acid sequence using GAP Weight of 8 and LengthWeight of 2, and the BLOSUM62 scoring matrix; or any equivalent programthereof. Anequivalent program is any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

The NELL1 peptide may be made synthetically, i.e. from individual aminoacids, or semi-synthetically, i.e. from oligopeptide units or acombination of oligopeptide units and individual amino acids. Suitablemethods for synthesizing proteins are described by Stuart and Young in“Solid Phase Peptide Synthesis,” Second Edition, Pierce Chemical Company(1984), Solid Phase Peptide Synthesis, Methods Enzymol., 289, AcademicPress, Inc, New York (1997).

The NELL1 peptide may also be prepared by methods that are well known inthe art. One such method includes isolating or synthesizing DNA encodingthe NELL1 peptide, and producing the recombinant protein by expressingthe DNA, optionally in a recombinant vector, in a suitable host cell.Suitable methods for synthesizing DNA are described by Caruthers et al.(1985) Science 230:281-285; and DNA Structure, Part A: Synthesis andPhysical Analysis of DNA, Lilley, D. M. J. and Dahlberg, J. E. (Eds.),Methods Enzymol., 211, Academic Press, Inc., New York (1992).

In some embodiments of the presently disclosed methods, a nucleic acidmolecule encoding a NELL1 peptide is administered to a subject in needthereof in order to regenerate tissue, promote the healing of an injury(e.g., tendon or ligament injury), or to enhance migration and/orproliferation of fibroblasts. As used herein, the terms “encoding” or“encoded” when used in the context of a specified nucleic acid mean thatthe nucleic acid comprises the requisite information to directtranslation of the nucleotide sequence into a specified polypeptide.

In some embodiments of the presently disclosed methods, the NELL1nucleic acid molecule is operably linked to at least one regulatoryelement. A regulatory element is a nucleic acid sequence(s) capable ofeffecting the expression of nucleic acid(s), or the peptide or proteinproduct thereof. Non-limiting examples of regulatory elements includepromoters, enhancers, polyadenylation signals, transcription ortranslation termination signals, ribosome binding sites, or othersegments of DNA where regulatory proteins, such as, but not limited to,transcription factors, bind preferentially to control gene expressionand thus protein expression.

Regulatory elements may be operably linked to the nucleic acids,peptides, or proteins of the described invention. When two or moreelements are operably linked, there exists a functional linkage betweenthe elements. For example, when a promoter and a protein coding sequenceare operably linked, the promoter sequence initiates and mediatestranscription of the protein coding sequence. The regulatory elementsneed not be contiguous with the nucleic acids, peptides, or proteinswhose expression they control as long as they function to direct theexpression thereof. Thus, for example, intervening untranslated yettranscribed sequences may be present between a promoter sequence and anucleic acid of the described invention and the promoter sequence maystill be considered operably linked to the coding sequence.

In certain embodiments, the NELL1 nucleic acid molecule is a recombinantexpression cassette or is part of an expression system. The term“recombinant expression cassette” refers to a nucleic acid construct,generated recombinantly or synthetically, with a series of specifiednucleic acid elements which permit transcription of a particular nucleicacid (e.g., protein coding sequence) in a host cell. The recombinantexpression cassette can be incorporated into a plasmid, chromosome,mitochondrial DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, apromoter, and a transcription termination signal such as a poly-Asignal.

The expression cassette or cloning vector can be generated usingmolecular biology techniques known in the art and utilizing restrictionenzymes, ligases, recombinases, and nucleic acid amplificationtechniques such as polymerase chain reaction that can be coupled withreverse transcription.

In some embodiments, the NELL1 protein is produced using a cell-freeexpression system such as the wheat germ in vitro translation system.

In some embodiments, the NELL1 nucleic acid molecule is in a host cellthat can be used for propagation of the nucleic acid molecule or forexpression of the NELL1 peptide and subsequent isolation and/orpurification. A host cell is any cell that contains a heterologousnucleic acid molecule. A heterologous polypeptide or nucleotide sequenceis a polypeptide or a sequence that originates from a different species,or if from the same species, is substantially modified from its nativeform in composition and/or genomic locus by deliberate humanintervention. The host cell typically supports the replication and/orexpression of the vector. Host cells may be prokaryotic cells such as,but not limited to, Escherichia coli, or eukaryotic cells such as, butnot limited to, yeast, insect, amphibian, plant (e.g., Nicotiana tabacum(tobacco), Oryza sativa (rice), Arabidopsis thaliana (cress)), ormammalian cells. The term as used herein means any cell which may existin culture or in vivo as part of a unicellular organism, part of amulticellular organism, or a fused or engineered cell culture. A cloninghost cell is a host cell that contains a cloning vector.

A recombinant cell or vector is one that has been modified by theintroduction of a heterologous nucleic acid or the cell that is derivedfrom a cell so modified. Recombinant cells express genes that are notfound in identical form within the native (non-recombinant) form of thecell or express native genes that are otherwise abnormally expressed,under-expressed or not expressed at all as a result of deliberate humanintervention. The alteration of a cell or vector by naturally occurringevents (e.g., spontaneous mutation, natural transformationtransduction/transposition), such as those occurring without deliberatehuman intervention, does not result in a recombinant cell or vector.

The NELL1 nucleic acid molecule can be introduced into a host cell forpropagation of production of NELL1 using any method known in the art,including transfection, transformation, or transduction, so long as thenucleic acid molecule gains access to the interior of the cell. Theinsertion or introduction of a nucleic acid into a cell refers totransfection or transformation or transduction and includes theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell(e.g., chromosome, plasmid, plastid or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (e.g., transfectedmRNA).

The NELL1 nucleic acid molecule can be introduced to allow for stabletransformation or transient transformation. Stable transformation isintended to mean that the nucleotide construct introduced into a cellintegrates into a genome of the cell. Transient transformation isintended to mean that a polynucleotide is introduced into the cell anddoes not integrate into a genome of the cell.

The NELL1 protein can be administered by a cell based gene therapy. Forexample, autologous, allogeneic or xenogeneic donor cells aregenetically modified in vitro to express and secrete NELL1 protein. Thegenetically modified donor cells are then subsequently implanted intothe subject in need of delivery of NELL1 protein in vivo. Examples ofsuitable cells include, but are not limited to, tenocytes, endothelialcells, fibroblasts (including tendon-derived fibroblasts), orstem/precursor cells, such as adult stem cells, embryonic stem cells,cord blood stem cells, perivascular stem cells, or tendon/stemprogenitor cells (TSPCs).

III. Methods

The presently disclosed methods involve the regeneration of tissue,promotion of healing of an injury (e.g., tendon or ligament injury), orthe enhancement of fibroblast migration and/or proliferation in asubject in need thereof. NELL1 peptides also find use in enhancingfibroblast migration and/or proliferation in an in vitro or ex vivosetting, for example in a wound scratch assay.

The regeneration of tissue refers to the process of renewal and growthof cells and extracellular matrix components within a particular tissuethat results in the production of tissue that has a cellular componentand architecture that allows for the normal functions of the particulartissue type. As described elsewhere herein, NELL1 has been demonstratedto stimulate the regeneration of tissues such as bone, cartilage, andskeletal muscle, which is believed to be attributed to at least one ofthe following effects of NELL1 protein: stimulation of thedifferentiation of precursor cells to maturity, pro-angiogenic activity,stimulation of ECM production, and reduction in levels of MMPs andinflammatory mediators. As disclosed herein, NELL1 also enhances themigration and/or proliferation of fibroblast cells, which can alsocontribute to tissue regeneration, particularly in response to a woundor within an area of injury.

As disclosed herein, the administration of NELL1 to fibroblast cellsresults in an increased migration rate and greater cell numbers withinan area of tissue injury. The greater cell numbers can be a result of anenhanced migration rate and/or an increase in proliferation of thefibroblasts. Thus, a NELL1 protein can enhance the migration and/orproliferation of fibroblast cells. An enhancement of fibroblastmigration refers to an increase in the rate of movement of fibroblastsfrom one region to another, for example, chemotactic movement towards achemical signal or movement towards an area of injury (i.e., a wound).Thus, NELL1 proteins find use in methods of enhancing the migrationand/or proliferation of fibroblast cells, in vivo or in vitro. In vitrosettings in which NELL1 proteins can enhance the migration and/orproliferation of fibroblast cells include wound scratch assays whereby aconfluent or substantially confluent layer of fibroblast cells grown inculture are disturbed through the introduction of a “scratch” or removalof a portion of the fibroblast monolayer, followed by a period ofculturing the cells such that the fibroblasts begin to repopulate the“wounded” area either through migration of the fibroblasts into thewounded area or proliferation of the fibroblasts, or a combination ofthe two.

The promotion of the healing of a wound refers to an increase in thespeed with which an injury (i.e., wound) heals or an improved outcome.Healing of an injury is considered to be promoted, for example, if thetime of healing of an injury treated with NELL1 compared to an injurynot treated with NELL1 is decreased by about 10%, about 25%, about 50%,or about 75%. Alternatively, healing of an injury is considered to bepromoted if the extent of re-acquisition of tendon and/or ligamentfunction of an injury treated with NELL1 compared to an injury nottreated with NELL1 is improved by about 10%, about 25%, about 50%, orabout 75%. Conversely, healing of an injury is considered to be promotedif the degree and/or amount of scar tissue resulting from an injurytreated with NELL1 as compared to an injury not treated with NELL1 isreduced by about 10%, about 25%, about 50%, or about 75%. Promotion ofhealing of an injury to a tendon or ligament can also be considered ifthere is an improvement in the composition (e.g., proportion and amountof collagen types) and/or architecture (e.g., correct alignment ofcollagen fibrils/fibers/bundles) of at least about 10%, about 25%, about50%, or about 75% of an injury treated with NELL1 compared to an injurynot treated with NELL1.

The NELL1 peptide or nucleic acid molecule encoding the same isadministered to a subject in need thereof to regenerate tissue,particularly to promote healing of an injury (e.g., a tendon and/orligament injury), or to enhance the migration and/or proliferation offibroblasts (e.g., dermal fibroblasts, ligament fibroblasts). The terms“subject”, “individual”, and “patient” are used interchangeably to referto a member of a species that comprises tendons and ligaments. Incertain embodiments, the subject is a mammal, including but not limitedto, mouse, rat, cat, goat, sheep, horse, hamster, ferret, pig, dog,platypus, guinea pig, rabbit and a primate, such as, for example, amonkey, ape, or human. In some of these embodiments, the subject is ahuman or a horse, such as a racehorse. Subjects in need of treatmentwith a NELL1 peptide or nucleic acid molecule include those having aninjury or those that are prone to injuries or an impaired healingprocess. Subjects that are prone to the development of injuries to aligament and/or tendon include racehorses and athletes. Subjects in needof treatment with a NELL1 peptide or nucleic acid molecular encoding thesame include those that have or are prone to impaired wound healing,impaired neovascularization, or impaired angiogenesis, including but notlimited to those subjects with diabetes (type 1 or type 2), vasculardiseases, hypercholesterolemia, and aging.

An injury or wound refers to damage or harm to a structure or functionof the body caused by intrinsic and/or extrinsic factors. Non-limitingintrinsic or extrinsic factors that can cause an injury or wound includethose of chemical, mechanical, thermal, bacterial, or physical means andencompass those that occur as the result of surgical procedures,overuse, or environmental conditions. The wound can be an open wound inwhich the skin is broken (e.g., lacerations, abrasions, puncture wound)or a closed wound. Particular wounds that can be healed with NELL1include, but are not limited to, bone injuries (e.g., complete orpartial fractures), skin wounds, and skeletal muscle injuries.

Intrinsic factors that can contribute to the development of injuries totendons and/or ligaments include genetic susceptibility, overuse, poorbiomechanics, poor nutrition, and obesity. The extrinsic factors areoften related to sports and include excessive forces or loading, poortraining techniques, environmental conditions, and surgical procedures.The injury to the tendon and/or ligament can be a closed wound or anopen wound, where the skin is lacerated, cut or punctured. The injurycan include inflammation, a sprain, strain, tearing, stretching, orlaceration of the tendon or ligament.

A tendon is a band of connective tissue that connects muscles to bonesor cartilage. A ligament is a band of connective tissue that connectsbones to other bones to form joints.

Injuries to tendons include tendinitis (acute tendon injury accompaniedby inflammation), tendinosis (chronic tendon injury with degeneration atthe cellular level and no inflammation), and other tendinopathiesexhibiting chronic tendon injury with no etiological implications. Withtendinosis, damage to collagen, cells, and the vascular components ofthe tendon can occur, such as irregularities of collagen fibrils (e.g.,disorientation, degeneration, thinning, non-uniformity in length ordiameter, increase in the amount of glycosaminoglycans between thefibrils), rounded tenocytes or other cell abnormalities, and theingrowth of blood vessels.

The healing of an injury to any type of tendon can be promoted withNELL1, including a hand flexor tendon, a tendon within the rotator cuff,and an Achilles tendon, and within horses, a superficial digital flexortendon (SDFT) and a deep digital flexor tendon (DDFT) of either thehindlimb(s) or forelimb(s).

Likewise, the healing of an injury to any type of ligament can bepromoted with NELL1, including an anterior cruciate ligament (ACL),posterior cruciate ligament (PCL), lateral collateral ligament (LCL),medial collateral ligament (MCL), and in horses, a suspensory ligamentof either the hindlimb(s) or forelimb(s). A common ligament injury inhorses that can be healed according to the presently disclosed methodsis proximal suspensory desmitis, an inflammation of the suspensoryligament just below the hock.

The NELL1 peptide or nucleic acid encoding the same can be administeredto subjects in need thereof in the form of a composition furthercomprising a carrier. The term “carrier” as used herein describes amaterial that does not cause significant irritation to an organism anddoes not abrogate the biological activity and properties of thecomposition of the described invention. Carriers must be of sufficientlyhigh purity and of sufficiently low toxicity to render them suitable foradministration to a subject being treated. The carrier can be inert, orit can possess pharmaceutical benefits.

A pharmaceutical composition is a composition that is employed toprevent, reduce in intensity, cure or otherwise treat a target conditionor disease.

A pharmaceutically acceptable carrier refers to one or more compatiblesolid or liquid filler, diluents or encapsulating substances which aresuitable for administration to a human or other vertebrate animal.

The formulations may be presented conveniently in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing into association the NELL1peptide or nucleic acid encoding the same (“active compound”) with thecarrier which constitutes one or more accessory agents. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active agent with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

The NELL1 peptide or nucleic acid encoding the same may be mixed withother active materials that do not impair the desired action, or withmaterials that supplement the desired action. Solutions or suspensionsused for parenteral, intradermal, subcutaneous, intrathecal, or topicalapplication may include, but are not limited to, for example, thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation may be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. Administeredintravenously, particular carriers are physiological saline or phosphatebuffered saline (PBS).

Pharmaceutical compositions for parenteral injection comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil) and injectable organic esters such asethyl oleate. Proper fluidity may be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.

These compositions also may contain adjuvants including preservativeagents, wetting agents, emulsifying agents, and dispersing agents.Prevention of the action of microorganisms may be ensured by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. It also may bedesirable to include isotonic agents, for example, sugars, sodiumchloride and the like. Prolonged absorption of the injectablepharmaceutical form may be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Suspensions, in addition to the active compounds, may contain suspendingagents, as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, andmixtures thereof.

Injectable depot forms are made by forming microencapsulated matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release may be controlled.Such long acting formulations may be formulated with suitable polymericor hydrophobic materials (for example as an emulsion in an acceptableoil) or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations also are prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissues.

The locally injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions that may bedissolved or dispersed in sterile water or other sterile injectablemedium just prior to use. Injectable preparations, for example, sterileinjectable aqueous or oleaginous suspensions, may be formulatedaccording to the known art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation also may be asterile injectable solution, suspension or emulsion in a nontoxic,parenterally acceptable diluent or solvent such as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P. and isotonic sodiumchloride solution. In addition, sterile, fixed oils conventionally areemployed or as a solvent or suspending medium. For this purpose anybland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid are used inthe preparation of injectables.

Formulations for parenteral (including but not limited to, subcutaneous,intradermal, intramuscular, intravenous, intrathecal and intraarticular)administration include aqueous and non-aqueous sterile injectionsolutions that may contain anti-oxidants, buffers, bacteriostats andsolutes, which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensions,which may include suspending agents and thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample sealed ampules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring the addition of the sterile liquidcarrier, for example, saline, water-for-injection, a semi-liquid foam,or gel, immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described. Alternatively, a NELL1 peptide ornucleic acid encoding the same is dissolved in a buffered liquidsolution that is frozen in a unit-dose or multi-dose container and laterthawed for injection or kept/stabilized under refrigeration until use.Any label on, or associated with, the container(s) indicates that theenclosed composition is used for promoting the healing of injuries totendons and/or ligaments.

The therapeutic agent(s) may be contained in controlled release systems.In order to prolong the effect of a drug, it often is desirable to slowthe absorption of the drug from subcutaneous, intrathecal, orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle. In some embodiments, the use of a long-termsustained release implant may be particularly suitable for treatment ofchronic conditions. Long-term sustained release implants are well-knownto those of ordinary skill in the art and include some of the releasesystems described above.

In some embodiments wherein the pharmaceutical composition is in theform of an implant, the NELL1 peptide or a nucleic acid encoding thesame is impregnated into drug eluting devices, scaffolds or matricesthat are implanted into an injured area to deliver NELL1 in a controlledrelease fashion. The protein can also be linked to sutures that are usedfor tendon and/or ligament surgeries. In those instances wherein theNELL1 peptide is delivered by genetically modified donor cells, thecells can be incorporated into a matrix containing an appropriatemicroenvironment to maintain, for a given time, the viability and growthof the genetically modified donor cells.

Non-limiting examples of suitable matrices include, but are not limitedto, wound dressings, collagen matrix, patches, and hydrogels. The matrixcan be applied to the injured tendon and/or ligament that has beenexposed post-surgically, for example. After the injured tendon and/orligament is healed, the matrix can be removed or the matrixincorporating the NELL1 peptide or nucleic acid encoding the same can bereplaced intermittently throughout the healing process. In someembodiments, a rapidly degradable (e.g., 3-5 days in horses and 1-2weeks in rats) scaffold or dressing is used to deliver NELL1 (e.g.,calcium alginate). Rapidly degradable scaffolds or dressings allow forthe release of a burst of NELL1 in the first phase of healing andactivates tissue regeneration instead of scarring pathways. In certainembodiments, the scaffold or dressing is simpler (e.g., consistingessentially of collagen type A), rather than a complex biologicalcarrier, such as those made from urinary bladder or intestinal liningsthat may comprise various growth factors and collagens. In someembodiments, the wound dressing or matrix used to deliver NELL1comprises or consists essentially of calcium alginate.

The NELL1 peptide or nucleic acid encoding the same can be administeredto a subject by dispensing, supplying, applying, or giving the NELL1peptide or nucleic acid encoding the same to the subject. Administrationmay be in vivo or administration directly to tissue ex vivo. Generally,NELL1 peptides, nucleic acid molecules encoding the same, orcompositions comprising the NELL1 peptide or nucleic acid may beadministered systemically either orally, buccally, parenterally,topically, by inhalation or insufflation (i.e., through the mouth orthrough the nose), or rectally in dosage unit formulations, optionallycontaining the conventional nontoxic pharmaceutically acceptablecarriers, adjuvants, and vehicles as desired, or may be locallyadministered by means such as, but not limited to, injection,implantation, grafting, or topical application. Additionaladministration may be performed, for example, intravenously,transmucosally, transdermally, intramuscularly, subcutaneously,intraperitoneally, intrathecally, intralymphatically, intralesionally,or epidurally.

Any suitable route of administration may be used to deliver the NELL1peptide or nucleic acid molecule encoding the same for the purposes oftissue regeneration, such as promoting the healing of an injury to atendon and/or ligament. In certain embodiments, the NELL1 peptide ornucleic acid encoding the same is administered locally to the site ofinjury or of desired tissue regeneration or fibroblast migration and/orproliferation. In some of these embodiments, the NELL1 peptide, NELL1nucleic acid molecule, or a composition comprising the NELL1 peptide orNELL1 nucleic acid molecule are administered parenterally. The term“parenteral” as used herein refers to introduction into the body by wayof an injection (i.e., administration by injection), including, forexample, subcutaneously (i.e., an injection beneath the skin beneath thedermis into the subcutaneous tissue or “superficial fascia”),intramuscularly (i.e., an injection into a muscle), intravenously (i.e.,an injection into a vein), intrathecally (i.e., an injection into thespace around the spinal cord or under the arachnoid membrane of thebrain), intrasternal injection or infusion techniques. A parenterallyadministered composition is delivered using a needle, e.g., a surgicalneedle. Injectable preparations, such as sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents.According to some such embodiments, the NELL1 peptide or nucleic acidmolecule encoding the same is administered by injection.

In certain embodiments, the NELL1 peptide or nucleic acid molecule isadministered as a spray onto a tissue, such as a tendon or ligament thathas been exposed surgically (e.g., tendon splitting procedures to easestrain). The NELL1 peptide or nucleic acid molecule can also beadministered via adhesion to novel materials such as nanoparticles.Lyophilized NELL protein, which may or not be reconstituted as a liquidor a gel, can be placed directly onto an injured tendon or ligament.

Administering can be performed, for example, once, a plurality of times,and/or over one or more extended periods. Generally, an effective doseof the NELL1 peptide or nucleic acid encoding the same is administeredto a subject one or more times. In certain preferred embodiments, thecourse of treatment will comprise multiple doses of the NELL1 peptide ornucleic acid encoding the same over a period of weeks or months. Morespecifically, the NELL1 peptide or nucleic acid encoding the same may beadministered once every day, every two days, every three days, everyfour days, every five days, every six days, every week, every ten days,every two weeks, every three weeks, every month, every six weeks, everytwo months, every ten weeks or every three months. In this regard, itwill be appreciated that the dosages may be altered or the interval maybe adjusted based on patient response and clinical practices.

An effective amount of a pharmaceutical composition of the invention isany amount that is effective to achieve its purpose. The effectiveamount, usually expressed in mg/kg can be determined by routine methodsduring pre-clinical and clinical trials by those of skill in the art.

The NELL1 peptide or nucleic acid encoding the same can be administeredimmediately after the injury to a tissue (e.g., the tendon and/orligament) occurred or the administration can be delayed post-injury forabout three hours, 12 hours, one day, two days, three days, four days,five days, six days, seven days, eight days, nine days, ten days, twoweeks, three weeks, or longer.

The NELL1 peptide or nucleic acid encoding the same can be administeredprior to, along with, or subsequent to another treatment for healing theinjury (e.g., tendon and/or ligament injury). Non-limiting examples ofother treatments include surgery, rehabilitation, cryotherapy,administration of precursor cells, extracellular matrix materials(synthetic or purified), anti-inflammatory agents, and analgesics.

NELL1 can be combined with cells that are important in the formation ofnew tissues. For example, for tendons and ligaments tenocytes,tendon-derived fibroblasts, tendon stem/progenitor cells (TSPCs) andperivascular stem cells, as well as adult stem cells, such asmesenchymal stem cells, adipose derived stem cells, and bone marrowaspirate, can be used. The combination of NELL1 with cells can bedelivered as an injectable mixture or in a complex scaffold (syntheticor natural scaffold) that degrades in the injured site and acts both asa starting architectural guide for new tissue to form and also forreleasing correct signals that draw into the injured site growth factorsand cells needed for healing. This can be either novel scaffolds orexisting commercial products (e.g. mesenchymal stem cells) that havealready demonstrated activity in healing injuries, such as those totendons and/or ligaments but whose activity can be boosted by theaddition of NELL1 for more severe or challenging traumatic tissuedamage. (Yanming et al. 2007; Tempfer et al. 2009; Nemoto et al. 2013)

NELL1 can be added to formulations or products that are acellularextracellular matrix materials either extracted from natural sources(e.g. linings of urinary bladder, small intestinal submucosa etc.) ormanufactured as a synthetic. Acellular products for regenerativemedicine that contain extracellular matrix material may not have all theneeded signals for tissue regeneration and the addition of NELL1 canenhance the ability of some of these materials to effect celldifferentiation and tissue maturation.

In practicing combination therapy, the NELL1 peptide or nucleic acidencoding the same and the additional treatment or therapeutic agent maybe administered to the subject simultaneously, either in a singlecomposition, or as two or more distinct compositions using the same ordifferent administration routes. Alternatively, the NELL1 peptide ornucleic acid encoding the same may precede, or follow, the additionaltreatment or therapeutic agent by, e.g., intervals ranging from minutesto weeks. In at least one embodiment, the NELL1 peptide or nucleic acidencoding the same and the additional treatment or therapeutic agent areadministered within about 5 minutes to about two weeks of each other. Inyet other embodiments, several days (2, 3, 4, 5, 6 or 7), several weeks(1, 2, 3, 4, 5, 6, 7 or 8) or several months (1, 2, 3, 4, 5, 6, 7 or 8)may lapse between administration of the NELL1 peptide or nucleic acidencoding the same and the additional treatment or therapeutic agent. Insome of these embodiments, the NELL1 peptide or nucleic acid isadministered along with platelet derived plasma (PRP).

IV. Articles of Manufacture

The invention includes kits comprising a NELL1 peptide or nucleic acidencoding the same comprising one or more containers. The kit can containa unit dosage of a NELL1 peptide or nucleic acid encoding the same, andmay also contain one or more additional agents, such as those agentsthat promote wound healing, stimulate the regeneration of a tissue, orpromote the migration or proliferation of fibroblast cells.

The components of the kit may be provided in one or more liquidsolutions, such as a sterile aqueous solution. Alternatively, thecomponents of the kit may be provided in a lyophilized form that issuitable for reconstitution with an aqueous or non-aqueous liquid. Sucha liquid may be provided in a separate container.

The kit can further comprise a label or package insert associated withthe container(s) providing information regarding the use of the kit,such as for regenerating tissues, promoting the healing of wounds, orpromoting the migration or proliferation of fibroblast cells.

IV. Miscellaneous

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Morespecifically, as used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aprotein” includes a plurality of proteins; reference to “a cell”includes mixtures of cells, and the like. In addition, ranges providedin the specification and appended claims include both end points and allpoints between the end points. Therefore, a range of 2.0 to 3.0 includes2.0, 3.0, and all points between 2.0 and 3.0.

Throughout this specification and the claims, the words “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise.

As used herein, the term “about,” when referring to a value is meant toencompass variations of, in some embodiments ±50%, in some embodiments±20%, in some embodiments ±10%, in some embodiments ±5%, in someembodiments ±1%, in some embodiments ±0.5%, and in some embodiments±0.1% from the specified amount, as such variations are appropriate toperform the disclosed methods or employ the disclosed compositions.

General methods in molecular genetics and genetic engineering useful inthe present invention are described in the current editions of MolecularCloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring HarborLaboratory Press), Gene Expression Technology (Methods in Enzymology,Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego,Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P.Deutscher, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide toMethods and Applications (Innis, et al. 1990. Academic Press, San Diego,Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed.(R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), and Gene Transfer andExpression Protocols, pp. 109-128, ed. E. J. Murray, The Humana PressInc., Clifton, N. J.). Reagents, cloning vectors, and kits for geneticmanipulation are available from commercial vendors such as BioRad,Stratagene, Invitrogen, ClonTech and Sigma-Aldrich Co.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forexample, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference, regardless of whether thephrase “incorporated by reference” is or is not used in relation to theparticular reference. The foregoing detailed description and theexamples that follow have been given for clarity of understanding. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described. Variations obviousto one skilled in the art are included in the invention defined by theclaims. Any section headings used herein are for organizational purposesand are not to be construed as limiting the subject matter described.

V. Sequence Summary and Sequences

The following Table 1 provides a summary of the included sequences.

TABLE 1 Nucleotide and amino acid sequences disclosed herein. SEQ ID NO.Description 1 Homo sapiens NELL1 isoform 1 transcript variant(nucleotide) 2 Homo sapiens NELL1 isoform 1 (amino acid) 3 Homo sapiensNELL1 isoform 2 transcript variant (nucleotide) 4 Homo sapiens NELL1isoform 2 (amino acid) 5 Equus caballus NELL1 isoform 1 (nucleotide) 6Equus caballus NELL1 isoform 1 (amino acid) 7 Equus caballus NELL1isoform 2 (nucleotide) 8 Equus caballus NELL1 isoform 2 (amino acid) 9Mus musculus NELL1 (nucleotide) 10 Mus musculus NELL1 (amino acid) 11Rattus norvegicus NELL1 (nucleotide) 12 Rattus norvegicus NELL1 (aminoacid) 13 Felis catus NELL1 isoform 1 (amino acid) 14 Felis catus NELL1isoform 2 (amino acid) 15 Canis lupis familiaris NELL1 (amino acid) 16Ovis aries NELL1 (amino acid) 17 Homo sapiens NELL1 fragment (aminoacid) 18 Equus caballus NELL1 fragment (amino acid) 19 Bos taurus NELL1(amino acid)

Homo sapiens NELL1 isoform 1 nucleotide sequence (SEQ ID NO: 1) and translatedamino acid sequence (SEQ ID NO: 2) atatgcgagc gcagcacccg gcgctgccga gccacctccc ccgccgcccg ctagcaagtt 60 tggcggctcc aagccaggcg cgcctcagga tccaggctca tttgcttcca cctagcttcg 120 gtgccccctg ctaggcgggg accctcgaga gcg atg ccg atg gat ttg att tta 174                                      Met Pro Met Asp Leu Ile Leu gtt gtg tgg ttc tgt gtg tgc act gcc agg aca gtg gtg ggc ttt ggg 222 Val Val Trp Phe Cys Val Cys Thr Ala Arg Thr Val Val Gly Phe Gly atg gac cct gac ctt cag atg gat atc gtc acc gag ctt gac ctt gtg 270 Met Asp Pro Asp Leu Gln Met Asp Ile Val Thr Glu Leu Asp Leu Val aac acc acc ctt gga gtt gct cag gtg tct gga atg cac aat gcc agc 318 Asn Thr Thr Leu Gly Val Ala Gln Val Ser Gly Met His Asn Ala Ser aaa gca ttt tta ttt caa gac ata gaa aga gag atc cat gca gct cct 366 Lys Ala Phe Leu Phe Gln Asp Ile Glu Arg Glu Ile His Ala Ala Pro cat gtg agt gag aaa tta att cag ctg ttc cgg aac aag agt gaa ttc 414 His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe acc att ttg gcc act gta cag cag aag cca tcc act tca gga gtg ata 462 Thr Ile Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile ctg tcc att cga gaa ctg gag cac agc tat ttt gaa ctg gag agc agt 510 Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser ggc ctg agg gat gag att cgg tat cac tac ata cac aat ggg aag cca 558 Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Ile His Asn Gly Lys Pro agg aca gag gca ctt cct tac cgc atg gca gat gga caa tgg cac aag 606 Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys gtt gca ctg tca gtt agc gcc tct cat ctc ctg ctc cat gtc gac tgt 654 Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Val Asp Cys aac agg att tat gag cgt gtg ata gac cct cca gat acc aac ctt ccc 702 Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Asp Thr Asn Leu Pro cca gga atc aat tta tgg ctt ggc cag cgc aac caa aag cat ggc tta 750 Pro Gly Ile Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu ttc aaa ggg atc atc caa gat ggg aag atc atc ttt atg ccg aat gga 798 Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly tat ata aca cag tgt cca aat cta aat cac act tgc cca acc tgc agt 846 Tyr Ile Thr Gln Cys Pro Asn Leu Asn His Thr Cys Pro Thr Cys Ser gat ttc tta agc ctg gtg caa gga ata atg gat tta caa gag ctt ttg 894 Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu gcc aag atg act gca aaa cta aat tat gca gag aca aga ctt agt caa 942 Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Ser Gln ttg gaa aac tgt cat tgt gag aag act tgt caa gtg agt gga ctg ctc 990 Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu tat cga gat caa gac tct tgg gta gat ggt gac cat tgc agg aac tgc 1038 Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys act tgc aaa agt ggt gcc gtg gaa tgc cga agg atg tcc tgt ccc cct 1086 Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro ctc aat tgc tcc cca gac tcc ctc cca gtg cac att gct ggc cag tgc 1134 Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ala Gly Gln Cys tgt aag gtc tgc cga cca aaa tgt atc tat gga gga aaa gtt ctt gca 1182 Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala gaa ggc cag cgg att tta acc aag agc tgt cgg gaa tgc cga ggt gga 1230 Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu Cys Arg Gly Gly gtt tta gta aaa att aca gaa atg tgt cct cct ttg aac tgc tca gaa 1278 Val Leu Val Lys Ile Thr Glu Met Cys Pro Pro Leu Asn Cys Ser Glu aag gat cac att ctt cct gag aat cag tgc tgc cgt gtc tgt aga ggt 1326 Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Arg Val Cys Arg Gly cat aac ttt tgt gca gaa gga cct aaa tgt ggt gaa aac tca gag tgc 1374 His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu Asn Ser Glu Cys aaa aac tgg aat aca aaa gct act tgt gag tgc aag agt ggt tac atc 1422 Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Ser Gly Tyr Ile tct gtc cag gga gac tct gcc tac tgt gaa gat att gat gag tgt gca 1470 Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala gct aag atg cat tac tgt cat gcc aat act gtg tgt gtc aac ctt cct 1518 Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro ggg tta tat cgc tgt gac tgt gtc cca gga tac att cgt gtg gat gac 1566 Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp ttc tct tgt aca gaa cac gat gaa tgt ggc agc ggc cag cac aac tgt 1614 Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly Gln His Asn Cys gat gag aat gcc atc tgc acc aac act gtc cag gga cac agc tgc acc 1662 Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr tgc aaa ccg ggc tac gtg ggg aac ggg acc atc tgc aga gct ttc tgt 1710 Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Arg Ala Phe Cys gaa gag ggc tgc aga tac ggt gga acg tgt gtg gct ccc aac aaa tgt 1758 Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys gtc tgt cca tct gga ttc aca gga agc cac tgc gag aaa gat att gat 1806 Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp gaa tgt tca gag gga atc att gag tgc cac aac cat tcc cgc tgc gtt 1854 Glu Cys Ser Glu Gly Ile Ile Glu Cys His Asn His Ser Arg Cys Val aac ctg cca ggg tgg tac cac tgt gag tgc aga agc ggt ttc cat gac 1902 Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp gat ggg acc tat tca ctg tcc ggg gag tcc tgt att gac att gat gaa 1950 Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu tgt gcc tta aga act cac acc tgt tgg aac gat tct gcc tgc atc aac 1998 Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn ctg gca ggg ggc ttt gac tgt ctc tgc ccc tct ggg ccc tcc tgc tct 2046 Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser ggt gac tgt cct cat gaa ggg ggg ctg aag cac aat ggc cag gtg tgg 2094 Gly Asp Cys Pro His Glu Gly Gly Leu Lys His Asn Gly Gln Val Trp acc ttg aaa gaa gac agg tgt tct gtc tgc tcc tgc aag gat ggc aag 2142 Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys ata ttc tgc cga cgg aca gct tgt gat tgc cag aat cca agt gct gac 2190 Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Ser Ala Asp cta ttc tgt tgc cca gaa tgt gac acc aga gtc aca agt caa tgt tta 2238 Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu gac caa aat ggt cac aag ctg tat cga agt gga gac aat tgg acc cat 2286 Asp Gln Asn Gly His Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His agc tgt cag cag tgt cgg tgt ctg gaa gga gag gta gat tgc tgg cca 2334 Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Val Asp Cys Trp Pro ctc act tgc ccc aac ttg agc tgt gag tat aca gct atc tta gaa ggg 2382 Leu Thr Cys Pro Asn Leu Ser Cys Glu Tyr Thr Ala Ile Leu Glu Gly gaa tgt tgt ccc cgc tgt gtc agt gac ccc tgc cta gct gat aac atc 2430 Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn Ile acc tat gac atc aga aaa act tgc ctg gac agc tat ggt gtt tca cgg 2478 Thr Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Tyr Gly Val Ser Arg ctt agt ggc tca gtg tgg acg atg gct gga tct ccc tgc aca acc tgt 2526 Leu Ser Gly Ser Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr Cysaaa tgc aag aat gga aga gtc tgt tgt tct gtg gat ttt gag tgt ctt 2574 Lys Cys Lys Asn Gly Arg Val Cys Cys Ser Val Asp Phe Glu Cys Leucaa aat aat tga agtatttaca gtggactcaa cgcagaagaa tggacgaaat 2626 Gln Asn Asn * gaccatccaa cgtgattaag gataggaatc ggtagtttgg tttttttgtt tgttttgttt 2686 ttttaaccac agataattgc caaagtttcc acctgaggac ggtgtttgga ggttgccttt 2746 tggacctacc actttgctca ttcttgctaa cctagtctag gtgacctaca gtgccgtgca 2806 tttaagtcaa tggttgttaa aagaagtttc ccgtgttgta aatcatgttt cccttatcag 2866 atcatttgca aatacattta aatgatctca tggtaaatgt tgatgtattt tttggtttat 2926 tttgtgtact aacataatag agagagactc agctcctttt atttattttg ttgatttatg 2986 gatcaaattc taaaataaag ttgcctgttg tgacttttgt cccatctact gcatacttag 3046 tgctgagatc cctgtaaaat gttttgatga aaatatgtat gtagagtcca gtcgcattat 3106 acatacattt catagtgctg aaccttctta aatgcctact cattcagctt aaacaggctg 3166 aagccaagta tgacaaagag gggaagggcc aaaaacataa tcaaagaata attttaaaga 3226 gaattcttgt ctctcttgca aaaaaaaaa 3255 Homo sapiens NELL1 isoform 1 amino acid sequence (SEQ ID NO: 2) Met Pro Met Asp Leu Ile Leu Val Val Trp Phe Cys Val Cys Thr Ala Arg Thr Val Val Gly Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile Val Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Ala Gln Val Ser Gly Met His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Ile Glu Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Ile Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Ile His Asn Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Val Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Asp Thr Asn Leu Pro Pro Gly Ile Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Tyr Ile Thr Gln Cys Pro Asn Leu Asn His Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Ser Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Met Cys Pro Pro Leu Asn Cys Ser Glu Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Arg Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Ser Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Arg Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ser Glu Gly Ile Ile Glu Cys His Asn His Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys His Asn Gly Gln Val Trp Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Ser Ala Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Asn Gly His Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Val Asp Cys Trp Pro Leu Thr Cys Pro Asn Leu Ser Cys Glu Tyr Thr Ala Ile Leu Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn Ile Thr Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Tyr Gly Val Ser Arg Leu Ser Gly Ser Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Arg Val Cys Cys Ser Val Asp Phe Glu Cys Leu Gln Asn Asn Homo sapiens NELL1 isoform 2 nucleotide sequence (SEQ ID NO: 3) and translatedamino acid sequence (SEQ ID NO: 4) atatgcgagc gcagcacccg gcgctgccga gccacctccc ccgccgcccg ctagcaagtt 60 tggcggctcc aagccaggcg cgcctcagga tccaggctca tttgcttcca cctagcttcg 120 gtgccccctg ctaggcgggg accctcgaga gcg atg ccg atg gat ttg att tta 174                                      Met Pro Met Asp Leu Ile Leu gtt gtg tgg ttc tgt gtg tgc act gcc agg aca gtg gtg ggc ttt ggg 222 Val Val Trp Phe Cys Val Cys Thr Ala Arg Thr Val Val Gly Phe Gly atg gac cct gac ctt cag atg gat atc gtc acc gag ctt gac ctt gtg 270 Met Asp Pro Asp Leu Gln Met Asp Ile Val Thr Glu Leu Asp Leu Val aac acc acc ctt gga gtt gct cag gtg tct gga atg cac aat gcc agc 318 Asn Thr Thr Leu Gly Val Ala Gln Val Ser Gly Met His Asn Ala Ser aaa gca ttt tta ttt caa gac ata gaa aga gag atc cat gca gct cct 366 Lys Ala Phe Leu Phe Gln Asp Ile Glu Arg Glu Ile His Ala Ala Pro cat gtg agt gag aaa tta att cag ctg ttc cgg aac aag agt gaa ttc 414 His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe acc att ttg gcc act gta cag cag aag cca tcc act tca gga gtg ata 462 Thr Ile Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile ctg tcc att cga gaa ctg gag cac agc tat ttt gaa ctg gag agc agt 510 Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser ggc ctg agg gat gag att cgg tat cac tac ata cac aat ggg aag cca 558 Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Ile His Asn Gly Lys Pro agg aca gag gca ctt cct tac cgc atg gca gat gga caa tgg cac aag 606 Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys gtt gca ctg tca gtt agc gcc tct cat ctc ctg ctc cat gtc gac tgt 654 Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Val Asp Cys aac agg att tat gag cgt gtg ata gac cct cca gat acc aac ctt ccc 702 Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Asp Thr Asn Leu Pro cca gga atc aat tta tgg ctt ggc cag cgc aac caa aag cat ggc tta 750 Pro Gly Ile Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu ttc aaa ggg atc atc caa gat ggg aag atc atc ttt atg ccg aat gga 798 Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly tat ata aca cag tgt cca aat cta aat cac act tgc cca acc tgc agt 846 Tyr Ile Thr Gln Cys Pro Asn Leu Asn His Thr Cys Pro Thr Cys Ser gat ttc tta agc ctg gtg caa gga ata atg gat tta caa gag ctt ttg 894 Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu gcc aag atg act gca aaa cta aat tat gca gag aca aga ctt agt caa 942 Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Ser Gln ttg gaa aac tgt cat tgt gag aag act tgt caa gtg agt gga ctg ctc 990 Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu tat cga gat caa gac tct tgg gta gat ggt gac cat tgc agg aac tgc 1038 Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys act tgc aaa agt ggt gcc gtg gaa tgc cga agg atg tcc tgt ccc cct 1086 Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro ctc aat tgc tcc cca gac tcc ctc cca gtg cac att gct ggc cag tgc 1134 Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ala Gly Gln Cys tgt aag gtc tgc cga cca aaa tgt atc tat gga gga aaa gtt ctt gca 1182 Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala gaa ggc cag cgg att tta acc aag agc tgt cgg gaa tgc cga ggt gga 1230 Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu Cys Arg Gly Gly gtt tta gta aaa att aca gaa atg tgt cct cct ttg aac tgc tca gaa 1278 Val Leu Val Lys Ile Thr Glu Met Cys Pro Pro Leu Asn Cys Ser Glu aag gat cac att ctt cct gag aat cag tgc tgc cgt gtc tgt aga ggt 1326 Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Arg Val Cys Arg Gly cat aac ttt tgt gca gaa gga cct aaa tgt ggt gaa aac tca gag tgc 1374 His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu Asn Ser Glu Cys aaa aac tgg aat aca aaa gct act tgt gag tgc aag agt ggt tac atc 1422 Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Ser Gly Tyr Ile tct gtc cag gga gac tct gcc tac tgt gaa gat att gat gag tgt gca 1470 Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala gct aag atg cat tac tgt cat gcc aat act gtg tgt gtc aac ctt cct 1518 Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro ggg tta tat cgc tgt gac tgt gtc cca gga tac att cgt gtg gat gac 1566 Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp ttc tct tgt aca gaa cac gat gaa tgt ggc agc ggc cag cac aac tgt 1614 Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly Gln His Asn Cys gat gag aat gcc atc tgc acc aac act gtc cag gga cac agc tgc acc 1662 Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr tgc aaa ccg ggc tac gtg ggg aac ggg acc atc tgc aga gct ttc tgt 1710 Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Arg Ala Phe Cys gaa gag ggc tgc aga tac ggt gga acg tgt gtg gct ccc aac aaa tgt 1758 Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys gtc tgt cca tct gga ttc aca gga agc cac tgc gag aaa gac att gat 1806 Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp gaa tgt gcc tta aga act cac acc tgt tgg aac gat tct gcc tgc atc 1854 Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile aac ctg gca ggg ggc ttt gac tgt cto tgc ccc tct ggg ccc tcc tgc 1902 Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys tct ggt gac tgt cct cat gaa ggg ggg ctg aag cac aat ggc cag gtg 1950 Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys His Asn Gly Gln Val tgg acc ttg aaa gaa gac agg tgt tct gtc tgc tcc tgc aag gat ggc 1998 Trp Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly aag ata ttc tgc cga cgg aca gct tgt gat tgc cag aat cca agt gct 2046 Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Ser Ala gac cta ttc tgt tgc cca gaa tgt gac acc aga gtc aca agt caa tgt 2094 Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys tta gac caa aat ggt cac aag ctg tat cga agt gga gac aat tgg acc 2142 Leu Asp Gln Asn Gly His Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr cat agc tgt cag cag tgt cgg tgt ctg gaa gga gag gta gat tgc tgg 2190 His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Val Asp Cys Trp cca cto act tgc ccc aac ttg agc tgt gag tat aca gct atc tta gaa 2238 Pro Leu Thr Cys Pro Asn Leu Ser Cys Glu Tyr Thr Ala Ile Leu Glu ggg gaa tgt tgt ccc cgc tgt gtc agt gac ccc tgc cta gct gat aac 2286 Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn atc acc tat gac atc aga aaa act tgc ctg gac agc tat ggt gtt tca 2334 Ile Thr Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Tyr Gly Val Ser cgg ctt agt ggc tca gtg tgg acg atg gct gga tct ccc tgc aca acc 2382 Arg Leu Ser Gly Ser Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr tgt aaa tgc aag aat gga aga gtc tgt tgt tct gtg gat ttt gag tgt 2430 Cys Lys Cys Lys Asn Gly Arg Val Cys Cys Ser Val Asp Phe Glu Cys ctt caa aat aat tga agtatttaca gtggactcaa cgcagaagaa tggacgaaat 2485 Leu Gln Asn Asn * gaccatccaa cgtgattaag gataggaatc ggtagtttgg tttttttgtt tgttttgttt 2545 ttttaaccac agataattgc caaagtttcc acctgaggac ggtgtttgga ggttgccttt 2605 tggacctacc actttgctca ttcttgctaa cctagtctag gtgacctaca gtgccgtgca 2665 tttaagtcaa tggttgttaa aagaagtttc ccgtgttgta aatcatgttt cccttatcag 2725 atcatttgca aatacattta aatgatctca tggtaaatgt tgatgtattt tttggtttat 2785 tttgtgtact aacataatag agagagactc agctcctttt atttattttg ttgatttatg 2845 gatcaaattc taaaataaag ttgcctgttg tgacttttgt cccatctact gcatacttag 2905 tgctgagatc cctgtaaaat gttttgatga aaatatgtat gtagagtcca gtcgcattat 2965 acatacattt catagtgctg aaccttctta aatgcctact cattcagctt aaacaggctg 3025 aagccaagta tgacaaagag gggaagggcc aaaaacataa tcaaagaata attttaaaga 3085 gaattcttgt ctctcttgca aaaaaaaaa 3114 Homo sapiens NELL1 isoform 2 amino acid sequence (SEQ ID NO: 4) Met Pro Met Asp Leu Ile Leu Val Val Trp Phe Cys Val Cys Thr Ala Arg Thr Val Val Gly Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile Val Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Ala Gln Val Ser Gly Met His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Ile Glu Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Ile Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Ile His Asn Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Val Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Asp Thr Asn Leu Pro Pro Gly Ile Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Tyr Ile Thr Gln Cys Pro Asn Leu Asn His Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Ser Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Met Cys Pro Pro Leu Asn Cys Ser Glu Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Arg Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Ser Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Arg Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys His Asn Gly Gln Val Trp Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Ser Ala Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Asn Gly His Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Val Asp Cys Trp Pro Leu Thr Cys Pro Asn Leu Ser Cys Glu Tyr Thr Ala Ile Leu Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn Ile Thr Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Tyr Gly Val Ser Arg Leu Ser Gly Ser Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Arg Val Cys Cys Ser Val Asp Phe Glu Cys Leu Gln Asn Asn Equus caballus NELL1 isoforrn 1 nucleotide sequence (SEQ ID NO: 5) andtranslated amino acid sequence (SEQ ID NO: 6) atg ggc ttt ggg atg gac ccc gac ctt caa atg gat att atc acc gag 48 Met Gly Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile Ile Thr Glu ctc gac ctc gtg aac acc acc ctt gga gtc act cag gtg tcc gga ctg 96 Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val Ser Gly Leu cac aat gcc agc aaa gca ttt tta ttt caa gat gta gag aga gag atc 144 His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Glu Arg Glu Ile cat gca gcc cca cac gtg agt gag aaa tta att cag ctg ttc cgg aat 192 His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn aag agt gaa ttc acc ttt ttg gcc act gtg cag cag aag ccg tca act 240 Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr tca gga gtg ata ctg tcc att cga gaa ctg gaa aac agt tat ttt gaa 288 Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu Asn Ser Tyr Phe Glu ctg gag agc agt ggc ctg aga gat gag att cga tat cac tac aca cac 336 Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Thr His aag ggg aag ccc agg aca gag gca ctt ccc tac cgg atg gcg gac gga 384 Lys Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly cgg tgg cac aag gtg gcg ctg tca gtt agc gcc tct cat ctc ctg ctc 432 Arg Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu cac atc gac tgc aac agg att tat gaa cgt gtg ata gac act cct gag 480 His Ile Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Thr Pro Glu acc aac ctc ccc cca gga agc aat ttg tgg ctg ggt cag cga aac caa 528 Thr Asn Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln Arg Asn Gln aag cac ggc tta ttc aaa gga atc atc caa gat gga aaa atc atc ttc 576 Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe atg ccg aat gga tac ata aca cag tgt ccg aac ctg aat cgc act tgc 624 Met Pro Asn Gly Tyr Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys cca acg tgc agt gat ttc tta agc ctg gtg caa gga atc atg gat tta 672 Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu caa gag ctt ctg gcc aag atg act gcg aaa cta aat tat gca gag aca 720 Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr cga ctt agt caa ttg gaa aac tgc cac tgc gag aag acc tgt caa gtg 768 Arg Leu Ser Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val agt gga ctg ctc tat aga gac cag gac tcc tgg gtt gat ggc gat cac 816 Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His tgt agg aac tgc acg tgc aaa agc ggc gct gtg gaa tgt cgg agg atg 864 Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met tct tgt ccc cct ctc aat tgc tcc cca gac tcc ctc cct gtg cac gtt 912 Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Val gcc ggc cag tgc tgt aag gtc tgc cga cca aaa tgt atc tac gga ggg 960 Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly aaa gtc ctt gca gaa ggc cag cgg att tta acc aag agc tgt cgg gaa 1008 Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu tgc cga ggt gga gtt tta gtg aaa att aca gaa gcg tgc cct cct ttg 1056 Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Pro Leu aac tgc tca gac aag gat cac att ctc cca gag aat cag tgc tgc agc 1104 Asn Cys Ser Asp Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Ser gtc tgc aga ggt cat aac ttt tgt gcg gaa gga cct aaa tgt ggt gaa 1152 Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu aat tca gag tgc aaa aac tgg aat aca aaa gct act tgc gag tgc aag 1200 Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys aat ggt tat atc tct gtc cag ggg gac tcc gcc tac tgt gaa gat atc 1248 Asn Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile gat gag tgt gct gct aag atg cat tac tgt cgt gcc aat act gtg tgt 1296 Asp Glu Cys Ala Ala Lys Met His Tyr Cys Arg Ala Asn Thr Val Cys gtc aac ctg cct ggg tta tat cgg tgt gac tgt gtc ccg gga tac att 1344 Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile cgc gtg gat gat ttc tct tgt aca gaa cat gac gaa tgt ggc agc ggg 1392 Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly cag cac aac tgt gat gag aat gcc atc tgc acc aac act gtc cag gga 1440 Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly cac agc tgc acc tgc aaa ccg ggc tac gtg ggg aat ggg acc agc tgc 1488 His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ser Cys aga gcg ttc tgc gaa gag ggc tgc aga tat ggc ggg aca tgc gtg gct 1536 Arg Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala cct aac aaa tgt gtc tgt cct tct gga ttc aca gga agc cac tgt gag 1584 Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu aaa gat att gat gaa tgt aca gag gga atc att gag tgc cac aac cat 1632 Lys Asp Ile Asp Glu Cys Thr Glu Gly Ile Ile Glu Cys His Asn His tcc cgc tgc gtt aac ctg cca ggg tgg tac cac tgt gag tgc aga agc 1680 Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser ggt ttc cat gac gat ggg acc tat tca ctg tcc ggg gag tcc tgt att 1728 Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile gac att gat gaa tgt gcc tta aga act cac acc tgt tgg aat gat tct 1776 Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser gcc tgc atc aac ttg gca ggg ggc ttc gac tgc ctg tgt ccc tca ggg 1824 Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly cca tcc tgc tct ggt gac tgc ccc cac gaa gga gga ctg aag cgc aac 1872 Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys Arg Asn ggg cag gtg tgg acc ctg aaa gaa gac agg tgt tct gtg tgt tcc tgc 1920 Gly Gln Val Trp Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser Cys aag gat ggg aag ata ttc tgc cga cgg aca gct tgt gat tgc cag aat 1968 Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn cca agc gtt gac ctt ttc tgt tgc cca gag tgt gac acc agg gtc aca 2016 Pro Ser Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr agt caa tgt tta gac caa aat gga cac aag ctc tat cga agt gga gac 2064 Ser Gln Cys Leu Asp Gln Asn Gly His Lys Leu Tyr Arg Ser Gly Asp aat tgg act cac agc tgt cag cag tgc cgg tgt ctg gaa gga gag gta 2112 Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Val gat tgc tgg cca ctc act tgc ccc aga ttg agc tgt gag tac aca gcc 2160 Asp Cys Trp Pro Leu Thr Cys Pro Arg Leu Ser Cys Glu Tyr Thr Ala atc ttg gaa ggg gag tgt tgt cca cgc tgt gtc agc gac ccc tgc ctg 2208 Ile Leu Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu gcg gat aac atc gtc tat gac atc aga gaa act tgc ctg gac agc tat 2256 Ala Asp Asn Ile Val Tyr Asp Ile Arg Glu Thr Cys Leu Asp Ser Tyr gga gtt tca agg ctt agt ggc tca gtg tgg aca ttg gct gga tct ccc 2304 Gly Val Ser Arg Leu Ser Gly Ser Val Trp Thr Leu Ala Gly Ser Pro tgc acg acc tgc aaa tgc aag aat gga agt gtc tgc tgt tct gtg gat 2352 Cys Thr Thr Cys Lys Cys Lys Asn Gly Ser Val Cys Cys Ser Val Asp ttg gag tgt ctt cat aat aat tga aggatttaaa atggactcat gatcgccaga 2406 Leu Glu Cys Leu His Asn Asn * gaaaaatgga caaatgacca tccatgatga tgaaagaaca ggagttggtg ttttttttac 2466 cacagacaat taccaaagtc tccgtctgag gaaggtgttt gcaggttgcc ttttggacct 2526 cccactctgc tcattcttgc taacctagtc taggtgacct acagtgcatt tcagtctatg 2586 gttgttaaaa gaagttttcc gtgttgtaaa tcacgtttcc cttaccaggt cattgcaaat 2646 acatttaaat gatttcatgg taaatgttga tgtatttttt gggtttattt tgtgtactaa 2706 cataatagag attcagctgc ttttatttat ttttttcttg acttttggat caaattcaac 2766 aaataaagtt gcctgttgtg atttt 2791 Equus caballus NELL1 isoform 1 amino acid sequence (SEQ ID NO: 6) Met Gly Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile Ile Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val Ser Gly Leu His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Glu Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu Asn Ser Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Thr His Lys Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Arg Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Ile Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Thr Pro Glu Thr Asn Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Tyr Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Ser Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Val Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Pro Leu Asn Cys Ser Asp Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Ser Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys Arg Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ser Cys Arg Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Thr Glu Gly Ile Ile Glu Cys His Asn His Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys Arg Asn Gly Gln Val Trp Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Ser Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Asn Gly His Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Val Asp Cys Trp Pro Leu Thr Cys Pro Arg Leu Ser Cys Glu Tyr Thr Ala Ile Leu Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn Ile Val Tyr Asp Ile Arg Glu Thr Cys Leu Asp Ser Tyr Gly Val Ser Arg Leu Ser Gly Ser Val Trp Thr Leu Ala Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Ser Val Cys Cys Ser Val Asp Leu Glu Cys Leu His Asn Asn Equus caballus NELL1 isoform 2 nucleotide sequence (SEQ ID NO: 7) and translatedamino acid sequence (SEQ ID NO: 8) atg ggc ttt ggg atg gac ccc gac ctt caa atg gat att atc acc gag 48 Met Gly Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile Ile Thr Glu ctc gac ctc gtg aac acc acc ctt gga gtc act cag gtg tcc gga ctg 96 Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val Ser Gly Leu cac aat gcc agc aaa gca ttt tta ttt caa gat gta gag aga gag atc 144 His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Glu Arg Glu Ile cat gca gcc cca cac gtg agt gag aaa tta att cag ctg ttc cgg aat 192 His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn aag agt gaa ttc acc ttt ttg gcc act gtg cag cag aag ccg tca act 240 Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr tca gga gtg ata ctg tcc att cga gaa ctg gaa aac agt tat ttt gaa 288 Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu Asn Ser Tyr Phe Glu ctg gag agc agt ggc ctg aga gat gag att cga tat cac tac aca cac 336 Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Thr His aag ggg aag ccc agg aca gag gca ctt ccc tac cgg atg gcg gac gga 384 Lys Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly cgg tgg cac aag gtg gcg ctg tca gtt agc gcc tct cat ctc ctg ctc 432 Arg Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu cac atc gac tgc aac agg att tat gaa cgt gtg ata gac act cct gag 480 His Ile Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Thr Pro Glu acc aac ctc ccc cca gga agc aat ttg tgg ctg ggt cag cga aac caa 528 Thr Asn Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln Arg Asn Gln aag cac ggc tta ttc aaa gga atc atc caa gat gga aaa atc atc ttc 576 Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe atg ccg aat gga tac ata aca cag tgt ccg aac ctg aat cgc act tgc 624 Met Pro Asn Gly Tyr Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys cca acg tgc agt gat ttc tta agc ctg gtg caa gga atc atg gat tta 672 Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu caa gag ctt ctg gcc aag atg act gcg aaa cta aat tat gca gag aca 720 Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr cga ctt agt caa ttg gaa aac tgc cac tgc gag aag acc tgt caa gtg 768 Arg Leu Ser Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val agt gga ctg cto tat aga gac cag gac tcc tgg gtt gat ggc gat cac 816 Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His tgt agg aac tgc acg tgc aaa agc ggc gct gtg gaa tgt cgg agg atg 864 Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met tct tgt ccc cct ctc aat tgc tcc cca gac tcc ctc cct gtg cac gtt 912 Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Val gcc ggc cag tgc tgt aag gtc tgc cga cca aaa tgt atc tac gga ggg 960 Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly aaa gtc ctt gca gaa ggc cag cgg att tta acc aag agc tgt cgg gaa 1008 Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu tgc cga ggt gga gtt tta gtg aaa att aca gaa gcg tgc cct cct ttg 1056 Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Pro Leu aac tgc tca gac aag gat cac att ctc cca gag aat cag tgc tgc agc 1104 Asn Cys Ser Asp Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Ser gtc tgc aga ggt cat aac ttt tgt gcg gaa gga cct aaa tgt ggt gaa 1152 Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu aat tca gag tgc aaa aac tgg aat aca aaa gct act tgc gag tgc aag 1200 Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys aat ggt tat atc tct gtc cag ggg gac tcc gcc tac tgt gaa gat atc 1248 Asn Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile gat gag tgt gct gct aag atg cat tac tgt cgt gcc aat act gtg tgt 1296 Asp Glu Cys Ala Ala Lys Met His Tyr Cys Arg Ala Asn Thr Val Cys gtc aac ctg cct ggg tta tat cgg tgt gac tgt gtc ccg gga tac att 1344 Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile cgc gtg gat gat ttc tct tgt aca gaa cat gac gaa tgt ggc agc ggg 1392 Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly cag cac aac tgt gat gag aat gcc atc tgc acc aac act gtc cag gga 1440 Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly cac agc tgc acc tgc aaa ccg ggc tac gtg ggg aat ggg acc agc tgc 1488 His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ser Cys aga gcg ttc tgc gaa gag ggc tgc aga tat ggc ggg aca tgc gtg gct 1536 Arg Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala cct aac aaa tgt gtc tgt cct tct gga ttc aca gga agc cac tgt gag 1584 Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu aaa gac att gat gaa tgt gcc tta aga act cac acc tgt tgg aat gat 1632 Lys Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp tct gcc tgc atc aac ttg gca ggg ggc ttc gac tgc ctg tgt ccc tca 1680 Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser ggg cca tcc tgc tct ggt gac tgc ccc cac gaa gga gga ctg aag cgc 1728 Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys Arg aac ggg cag gtg tgg acc ctg aaa gaa gac agg tgt tct gtg tgt tcc 1776 Asn Gly Gln Val Trp Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser tgc aag gat ggg aag ata ttc tgc cga cgg aca gct tgt gat tgc cag 1824 Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln aat cca agc gtt gac ctt ttc tgt tgc cca gag tgt gac acc agg gtc 1872 Asn Pro Ser Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val aca agt caa tgt tta gac caa aat gga cac aag ctc tat cga agt gga 1920 Thr Ser Gln Cys Leu Asp Gln Asn Gly His Lys Leu Tyr Arg Ser Gly gac aat tgg act cac agc tgt cag cag tgc cgg tgt ctg gaa gga gag 1968 Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu gta gat tgc tgg cca ctc act tgc ccc aga ttg agc tgt gag tac aca 2016 Val Asp Cys Trp Pro Leu Thr Cys Pro Arg Leu Ser Cys Glu Tyr Thr gcc atc ttg gaa ggg gag tgt tgt cca cgc tgt gtc agc gac ccc tgc 2064 Ala Ile Leu Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys ctg gcg gat aac atc gtc tat gac atc aga gaa act tgc ctg gac agc 2112 Leu Ala Asp Asn Ile Val Tyr Asp Ile Arg Glu Thr Cys Leu Asp Ser tat gga gtt tca agg ctt agt ggc tca gtg tgg aca ttg gct gga tct 2160 Tyr Gly Val Ser Arg Leu Ser Gly Ser Val Trp Thr Leu Ala Gly Ser ccc tgc acg acc tgc aaa tgc aag aat gga agt gtc tgc tgt tct gtg 2208 Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Ser Val Cys Cys Ser Val gat ttg gag tgt ctt cat aat aat tga aggatttaaa atggactcat 2255 Asp Leu Glu Cys Leu His Asn Asn *  gatcgccaga gaaaaatgga caaatgacca2285  Equus caballus NELL1 isoform 2 amino acid sequence (SEQ ID NO: 8) Met Gly Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile Ile Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val Ser Gly Leu His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Glu Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu Asn Ser Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Thr His Lys Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Arg Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Ile Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Thr Pro Glu Thr Asn Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Tyr Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Ser Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Val Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Pro Leu Asn Cys Ser Asp Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Ser Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys Arg Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ser Cys Arg Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys Arg Asn Gly Gln Val Trp Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Ser Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Asn Gly His Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Val Asp Cys Trp Pro Leu Thr Cys Pro Arg Leu Ser Cys Glu Tyr Thr Ala Ile Leu Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn Ile Val Tyr Asp Ile Arg Glu Thr Cys Leu Asp Ser Tyr Gly Val Ser Arg Leu Ser Gly Ser Val Trp Thr Leu Ala Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Ser Val Cys Cys Ser Val Asp Leu Glu Cys Leu His Asn Asn Mus muculus NELL1 nucleotide sequence (SEQ ID NO: 9) and translatedamino acid sequence (SEQ ID NO: 10) gcgttggtgc gccctgcttg gcggggggcc tccggagcg atg ccg atg gat gtg 54                                            Met Pro Met Asp Val att tta gtt ttg tgg ttc tgt gtg tgc acc gcc agg aca gtg ctg ggc 102 Ile Leu Val Leu Trp Phe Cys Val Cys Thr Ala Arg Thr Val Leu Gly ttt ggg atg gac cct gac ctt cag atg gac atc atc act gaa ctt gac 150 Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile Ile Thr Glu Leu Asp ctt gtg aac acc acc ctg ggc gtc act cag gtg gct gga cta cac aat 198 Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val Ala Gly Leu His Asn gcc agt aag gca ttt ctg ttt caa gat gta cag aga gag atc cac tca 246 Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Gln Arg Glu Ile His Ser gcc cct cat gtg agt gag aag ctg atc cag cta ttc cgg aat aag agt 294 Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser gag ttt acc ttt ttg gct aca gtg cag cag aag ccg tcc acc tca ggg 342 Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly gtg ata ctg tcg atc cgg gag ctg gaa cac agc tat ttt gaa ctg gag 390 Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu agc agt ggc cca aga gaa gag ata cgc tat cat tac atc cat ggc ggc 438 Ser Ser Gly Pro Arg Glu Glu Ile Arg Tyr His Tyr Ile His Gly Gly aag ccc agg act gag gcc ctt ccc tac cgc atg gcc gat gga cag tgg 486 Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp cac aag gtc gcg ctg tct gtg agc gcc tct cac ctc cta ctc cat gtc 534 His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Val gac tgc aat agg att tat gag cgt gtg ata gat cct ccg gag acc aac 582 Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Glu Thr Asn ctt cct cca gga agc aat cta tgg ctt ggg caa cgt aat caa aag cat 630 Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His ggc ttt ttc aaa gga atc atc caa gat ggc aag atc atc ttc atg ccg 678 Gly Phe Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro aac ggc ttc atc aca cag tgc cco aac cta aat cgc act tgc cca aca 726 Asn Gly Phe Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys Pro Thr tgc agt gat ttc ctg agc ctg gtt caa gga ata atg gat ttg caa gag 774 Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu ctt ttg gcc aag atg act gca aaa ctg aat tat gca gag acg aga ctt 822 Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu ggt caa ctg gaa aat tgc cac tgt gag aag acc tgc caa gtg agt ggg 870 Gly Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly ctg ctc tac agg gac caa gac tcc tgg gta gat ggt gac aac tgc agg 918 Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp Asn Cys Arg aac tgc aca tgc aaa agt ggt gct gtg gag tgc cga agg atg tcc tgt 966 Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys ccc cca ctc aac tgt tcc cca gac tca ctt cct gtg cat att tct ggc 1014 Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ser Gly caa tgt tgt aaa gtt tgc aga cca aaa tgt atc tat gga gga aaa gtt 1062 Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val ctt gct gag ggc cag cgg att tta acc aag acc tgc cgg gaa tgt cga 1110 Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Thr Cys Arg Glu Cys Arg ggt gga gtc ttg gta aaa atc aca gaa gct tgc cct cct ttg aac tgc 1158 Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Pro Leu Asn Cys tca gag aag gat cat att ctt ccg gag aac cag tgc tgc agg gtc tgc 1206 Ser Glu Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Arg Val Cys cga ggt cat aac ttc tgt gca gaa gca cct aag tgt gga gaa aac tcg 1254 Arg Gly His Asn Phe Cys Ala Glu Ala Pro Lys Cys Gly Glu Asn Ser gaa tgc aaa aat tgg aat aca aaa gcg act tgt gag tgc aag aat gga 1302 Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly tac atc tct gtc cag ggc aac tct gca tac tgt gaa gat atc gat gag 1350 Tyr Ile Ser Val Gln Gly Asn Ser Ala Tyr Cys Glu Asp Ile Asp Glu tgt gca gca aag atg cac tac tgt cat gcc aac acg gtg tgt gtc aac 1398 Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn ttg ccg ggg tta tat cgc tgt gac tgc atc cca gga tac atc cgt gtg 1446 Leu Pro Gly Leu Tyr Arg Cys Asp Cys Ile Pro Gly Tyr Ile Arg Val gat gac ttc tct tgt acg gag cat gat gat tgt ggc agc gga caa cac 1494 Asp Asp Phe Ser Cys Thr Glu His Asp Asp Cys Gly Ser Gly Gln His aac tgt gac aaa aat gcc atc tgt acc aac aca gtc cag gga cac agc 1542 Asn Cys Asp Lys Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser tgt acc tgc cag cca ggc tac gtg gga aat ggt act gtc tgc aaa gca 1590 Cys Thr Cys Gln Pro Gly Tyr Val Gly Asn Gly Thr Val Cys Lys Ala ttc tgt gaa gag ggt tgc aga tac gga ggt acc tgt gtg gcc cct aac 1638 Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn aaa tgt gtc tgt cct tct gga ttc aca gga agc cac tgt gag aaa gat 1686 Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp att gat gaa tgt gca gag gga ttc gtt gag tgc cac aac cac tcc cgc 1734 Ile Asp Glu Cys Ala Glu Gly Phe Val Glu Cys His Asn His Ser Arg tgc gtt aac ctt cca ggg tgg tac cac tgt gag tgc aga agc ggt ttc 1782 Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe cat gac gat ggg acc tat tca ctg tcc ggg gag tcc tgc att gat att 1830 His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile gat gaa tgt gcc tta aga act cac act tgt tgg aat gac tct gcc tgc 1878 Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys atc aac tta gca gga gga ttt gac tgc ctg tgt ccc tct ggg ccc tcc 1926 Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser tgc tct ggt gac tgt ccc cac gaa ggg ggg ctg aag cat aat ggg cag 1974 Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys His Asn Gly Gln gtg tgg att ctg aga gaa gac agg tgt tca gtc tgt tcc tgt aag gat 2022 Val Trp Ile Leu Arg Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp ggg aag ata ttc tgc cgg cgg aca gct tgt gat tgc cag aat cca aat 2070 Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Asn gtt gac ctt ttc tgc tgc cca gag tgt gac acc agg gtc act agc caa 2118 Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln tgt tta gat caa agc gga cag aag ctc tat cga agt gga gac aac tgg 2166 Cys Leu Asp Gln Ser Gly Gln Lys Leu Tyr Arg Ser Gly Asp Asn Trp acc cac agc tgc cag cag tgc cga tgt ctg gaa gga gag gca gac tgc 2214 Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Ala Asp Cys tgg cct cta gct tgc cct agt ttg agc tgt gaa tac aca gcc atc ttt 2262 Trp Pro Leu Ala Cys Pro Ser Leu Ser Cys Glu Tyr Thr Ala Ile Phe gaa gga gag tgt tgt ccc cgc tgt gtc agt gac ccc tgc ctg gct gat 2310 Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp aat att gcc tat gac atc aga aaa act tgc ctg gac agc tct ggt att 2358 Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Ser Gly Ile tcg agg ctg agc ggc gca gtg tgg aca atg gct gga tct ccc tgt aca 2406 Ser Arg Leu Ser Gly Ala Val Trp Thr Met Ala Gly Ser Pro Cys Thr acc tgt caa tgc aag aat ggg aga gtc tgc tgc tct gtg gat ctg gtg 2454 Thr Cys Gln Cys Lys Asn Gly Arg Val Cys Cys Ser Val Asp Leu Val tgt ctt gag aat aac tga agattttaaa tggactcatc acatgagaaa 2502 Cys Leu Glu Asn Asn * atggacaaaa tgaccatcca acctgaggaa gaggaggggc tgatttcttt ttctttttaa 2562 ccacagtcaa ttaccaaagt ctccatcaga ggaaggcgtt tgggttgcct ttaccacttt 2622 gctcatcctt gctgacctag tctagatgcc tgcagtaccg tgtatttcgg tcgatggttg 2682 ttgagtctcc gtgctgtaaa tcacatttcc cttgtcagat catttacaga tacatttaaa 2742 ggattccatg ataaatgtta aagtaccttt tgtttatttt gtgtaccaac ataatagaga 2802 cttggcacca 2812  Mus musculus NELL1 amino acid sequence (SEQ ID NO: 10) Met Pro Met Asp Val Ile Leu Val Leu Trp Phe Cys Val Cys Thr Ala Arg Thr Val Leu Gly Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile Ile Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val Ala Gly Leu His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Gln Arg Glu Ile His Ser Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Pro Arg Glu Glu Ile Arg Tyr His Tyr Ile His Gly Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Val Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Glu Thr Asn Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Phe Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Phe Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Gly Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp Asn Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ser Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Thr Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Pro Leu Asn Cys Ser Glu Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Arg Val Cys Arg Gly His Asn Phe Cys Ala Glu Ala Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asn Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Ile Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Asp Cys Gly Ser Gly Gln His Asn Cys Asp Lys Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Gln Pro Gly Tyr Val Gly Asn Gly Thr Val Cys Lys Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ala Glu Gly Phe Val Glu Cys His Asn His Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys His Asn Gly Gln Val Trp Ile Leu Arg Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Asn Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Ser Gly Gln Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Ala Asp Cys Trp Pro Leu Ala Cys Pro Ser Leu Ser Cys Glu Tyr Thr Ala Ile Phe Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Ser Gly Ile Ser Arg Leu Ser Gly Ala Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr Cys Gln Cys Lys Asn Gly Arg Val Cys Cys Ser Val Asp Leu Val Cys Leu Glu Asn Asn Rattus norvegicus NELL1 nucleotide sequence (SEQ ID NO: 11) and translatedamino acid sequence (SEQ ID NO: 12) aagcactggt ttcttgttag cgttggtgcg ccctgcttgg cgggggttct ccggagcg 58 atg ccg atg gat gtg att tta gtt ttg tgg ttc tgt gta tgc acc gcc 106 Met Pro Met Asp Val Ile Leu Val Leu Trp Phe Cys Val Cys Thr Ala agg aca gtg ttg ggc ttt ggg atg gac cct gac ctt cag ctg gac atc 154 Arg Thr Val Leu Gly Phe Gly Met Asp Pro Asp Leu Gln Leu Asp Ile atc tca gag ctc gac ctg gtg aac acc acc ctg gga gtc acg cag gtg 202 Ile Ser Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val gct gga ctg cac aac gcc agt aaa gca ttt cta ttt caa gat gta cag 250 Ala Gly Leu His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Gln aga gag atc cat tcg gcc cct cac gtg agt gag aag ctg atc cag cta 298 Arg Glu Ile His Ser Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu ttc cgg aat aag agc gag ttc acc ttt ttg gct aca gtg cag cag aaa 346 Phe Arg Asn Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys cca tcc acc tca ggg gtg ata ctg tcc atc cgg gag ctg gag cac agc 394 Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser tat ttt gaa ctg gag agc agt ggc cca aga gaa gag ata cgc tac cat 442 Tyr Phe Glu Leu Glu Ser Ser Gly Pro Arg Glu Glu Ile Arg Tyr His tac ata cat ggt gga aag ccc agg act gag gcc ctt ccc tac cgc atg 490 Tyr Ile His Gly Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met gca gac gga caa tgg cac aag gtc gcg ctg tca gtg agc gcc tct cac 538 Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His ctc ctg ctc cac atc gac tgc aat agg att tac gag cgt gtg ata gac 586 Leu Leu Leu His Ile Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp cct ccg gag acc aac ctt cct cca gga agc aat ctg tgg ctt ggg caa 634 Pro Pro Glu Thr Asn Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln cgt aac caa aag cat ggc ttt ttc aaa gga atc atc caa gat ggt aag 682 Arg Asn Gln Lys His Gly Phe Phe Lys Gly Ile Ile Gln Asp Gly Lys atc atc ttc atg ccg aat ggt ttc atc aca cag tgt ccc aac ctc aat 730 Ile Ile Phe Met Pro Asn Gly Phe Ile Thr Gln Cys Pro Asn Leu Asn cgc act tgc cca aca tgc agt gac ttc ctg agc ctg gtt caa gga ata 778 Arg Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile atg gat ttg caa gag ctt ttg gcc aag atg act gca aaa ctg aat tat 826 Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr gca gag acg aga ctt ggt caa ctg gaa aat tgc cac tgt gag aag acc 874 Ala Glu Thr Arg Leu Gly Gln Leu Glu Asn Cys His Cys Glu Lys Thr tgc caa gtg agt ggg ctg ctc tac agg gac caa gac tcc tgg gtg gat 922 Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp ggt gac aac tgt ggg aac tgc acg tgc aaa agt ggt gcc gtg gag tgc 970 Gly Asp Asn Cys Gly Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys cgc agg atg tcc tgt ccc ccg ctc aac tgt tcc ccg gac tca ctt cct 1018 Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro gtg cac att tcc ggc cag tgt tgt aaa gtt tgc aga cca aaa tgt atc 1066 Val His Ile Ser Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile tat gga gga aaa gtt ctt gct gag ggc cag cgg att tta acc aag acc 1114 Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Thr tgc cgg gaa tgt cga ggt gga gtc ttg gta aaa atc aca gaa gct tgc 1162 Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys cct cct ttg aac tgc tca gca aag gat cat att ctt cca gag aat cag 1210 Pro Pro Leu Asn Cys Ser Ala Lys Asp His Ile Leu Pro Glu Asn Gln tgc tgc agg gtc tgc cca ggt cat aac ttc tgt gca gaa gca cct aag 1258 Cys Cys Arg Val Cys Pro Gly His Asn Phe Cys Ala Glu Ala Pro Lys tgc gga gaa aac tcg gaa tgc aaa aat tgg aat aca aaa gca acc tgt 1306 Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys gag tgc aag aat gga tac atc tct gtc cag ggc aac tct gca tac tgt 1354 Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asn Ser Ala Tyr Cys gaa gat att gat gag tgt gca gct aaa atg cac tat tgt cat gcc aac 1402 Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn acc gtg tgt gtc aac ttg ccg ggg ttg tat cgc tgt gac tgc gtc cca 1450 Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro ggg tac atc cgt gtg gat gac ttc tct tgt acg gag cat gat gat tgt 1498 Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Asp Cys ggc agc gga caa cac aac tgc gac aaa aat gcc atc tgt acc aac aca 1546 Gly Ser Gly Gln His Asn Cys Asp Lys Asn Ala Ile Cys Thr Asn Thr gtc cag gga cac agc tgc acc tgc cag ccg ggt tac gtg gga aat ggc 1594 Val Gln Gly His Ser Cys Thr Cys Gln Pro Gly Tyr Val Gly Asn Gly acc atc tgc aaa gca ttc tgt gaa gag ggt tgc aga tac gga ggt acc 1642 Thr Ile Cys Lys Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr tgt gtg gct cct aac aag tgt gtc tgt cct tct gga ttc acg gga agc 1690 Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser cac tgt gag aaa gat att gat gaa tgc gca gag gga ttc gtt gaa tgc 1738 His Cys Glu Lys Asp Ile Asp Glu Cys Ala Glu Gly Phe Val Glu Cys cac aac tac tcc cgc tgt gtt aac ctg cca ggg tgg tac cac tgt gag 1786 His Asn Tyr Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu tgc aga agc ggt ttc cat gac gat ggg acc tac tca ctg tcc ggg gag 1834 Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu tcc tgc att gat atc gat gaa tgt gcc tta aga act cac act tgt tgg 1882 Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp aat gac tct gcc tgc atc aac tta gca gga gga ttt gac tgc ctg tgt 1930 Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys ccc tct ggg ccc tcc tgc tct ggt gac tgt ccc cac gaa gga ggg ctg 1978 Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu aag cat aat ggg cag gtg tgg att ctg aga gaa gac agg tgt tca gtc 2026 Lys His Asn Gly Gln Val Trp Ile Leu Arg Glu Asp Arg Cys Ser Val tgt tcc tgc aag gat ggg aag ata ttc tgc cgg cgg aca gct tgt gat 2074 Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp tgc cag aat cca aat gtt gac ctt ttt tgc tgc cca gag tgc gat acc 2122 Cys Gln Asn Pro Asn Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr agg gtc acc agc caa tgt tta gat caa agt gga cag aag ctc tat cga 2170 Arg Val Thr Ser Gln Cys Leu Asp Gln Ser Gly Gln Lys Leu Tyr Arg agt gga gac aac tgg acc cac agc tgc cag cag tgc cga tgt ctg gaa 2218 Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu gga gag gca gac tgc tgg cct ctg gct tgc cct agt ttg ggc tgt gaa 2266 Gly Glu Ala Asp Cys Trp Pro Leu Ala Cys Pro Ser Leu Gly Cys Glu tac aca gcc atg ttt gaa ggg gag tgt tgt ccc cga tgt gtc agt gac 2314 Tyr Thr Ala Met Phe Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp ccc tgc ctg gct ggt aat att gcc tat gac atc aga aaa act tgc ctg 2362 Pro Cys Leu Ala Gly Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu gac agc ttt ggt gtt tcg agg ctg agc gga gcc gtg tgg aca atg gct 2410 Asp Ser Phe Gly Val Ser Arg Leu Ser Gly Ala Val Trp Thr Met Ala gga tct cct tgt aca acc tgc aaa tgc aag aat ggg aga gtc tgc tgc 2458 Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Arg Val Cys Cys tct gtg gat ctg gag tgt att gag aat aac tga agattttaaa tggactcgtc 2511 Ser Val Asp Leu Glu Cys Ile Glu Asn Asn * acgtgagaaa atgggcaaaa tgatcatccc acctgaggaa gaagaggggc tgatttcttt 2571 ttctttttaa ccacagtcaa ttaccaaagt ctccatctga ggaaggcgtt tggattgcct 2631 ttgccacttt gctcatcctt gctgacctag tctagatgcc tgcagtaccg tgcatttcgg 2691 tcgatggttg ttgagtctca gtgttgtaaa tcgcatttcc ctcgtcagat catttacaga 2751 tacatttaaa ggggttccat gataaatgtt aatgtaactt ttgtttattt tgtgtactga 2811 cataatagag acttggcacc atttatttat ttttcttgat ttttggatca aattctaaaa 2871 ataaagttgc ctgttgcgaa aaaaaaaaaa aaaaaaaaaa aaaa 2915 Rattus norvegicus NELL1 amino acid sequence (SEQ ID NO: 12) Met Pro Met Asp Val Ile Leu Val Leu Trp Phe Cys Val Cys Thr Ala Arg Thr Val Leu Gly Phe Gly Met Asp Pro Asp Leu Gln Leu Asp Ile Ile Ser Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val Ala Gly Leu His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Gln Arg Glu Ile His Ser Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Pro Arg Glu Glu Ile Arg Tyr His Tyr Ile His Gly Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Ile Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Glu Thr Asn Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Phe Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Phe Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Gly Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp Asn Cys Gly Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ser Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Thr Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Pro Leu Asn Cys Ser Ala Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Arg Val Cys Pro Gly His Asn Phe Cys Ala Glu Ala Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asn Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Asp Cys Gly Ser Gly Gln His Asn Cys Asp Lys Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Gln Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Lys Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ala Glu Gly Phe Val Glu Cys His Asn Tyr Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys His Asn Gly Gln Val Trp Ile Leu Arg Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Asn Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Ser Gly Gln Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Ala Asp Cys Trp Pro Leu Ala Cys Pro Ser Leu Gly Cys Glu Tyr Thr Ala Met Phe Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Gly Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Phe Gly Val Ser Arg Leu Ser Gly Ala Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Arg Val Cys Cys Ser Val Asp Leu Glu Cys Ile Glu Asn Asn Felis catus NELL1 isoform 1 amino acid sequence (SEQ ID NO: 13) Met Pro Arg Asp Val Ile Leu Val Val Trp Phe Cys Val Cys Thr Ala Arg Thr Val Val Gly Phe Gly Thr Asp Pro Asp Leu Gln Val Asp Ile Ile Ala Glu Leu Asp Leu Val Asn Thr Thr Ala Gly Val Thr Gln Val Ser Gly Leu His Asn Ala Ser Lys Ala Tyr Leu Phe Gln Glu Thr Glu Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Ser Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Ile His Asn Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Ile Ser Ala Ser His Leu Leu Leu His Val Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Glu Thr Asn Leu Pro Pro Gly Ser Asn Val Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Tyr Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Asn Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Asp Ala Cys Pro Pro Leu Asn Cys Ser Glu Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Ser Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Thr Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Arg Ala Phe Cys Gln Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ser Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Thr Glu Gly Ile Ile Glu Cys His Asn His Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys Arg Asn Gly Gln Val Trp Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Ser Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Asn Gly His Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Val Asp Cys Trp Pro Leu Thr Cys Pro Asn Leu Ser Cys Glu Tyr Thr Ala Met Leu Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Asp Ala Cys Pro Pro Leu Asn Cys Ser Glu Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Ser Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Thr Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Arg Ala Phe Cys Gln Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ser Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys Arg Asn Gly Gln Val Trp Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Ser Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Asn Gly His Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Val Asp Cys Trp Pro Leu Thr Cys Pro Asn Leu Ser Cys Glu Tyr Thr Ala Met Leu Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Tyr Gly Ile Ser Arg Leu Ser Gly Ala Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Ser Val Cys Cys Ser Val Asp Leu Glu Cys Leu His Asn Asn Felis catus NELL1 isoform 2 amino acid sequence (SEQ ID NO: 14)Met Pro Arg Asp Val Ile Leu Val Val Trp Phe Cys Val Cys Thr AlaArg Thr Val Leu Gly Phe Gly Met Asp Pro Asp Leu Gln Leu Asp Ile Ile Ser Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val Ala Gly Leu His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Gln Arg Glu Ile His Ser Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Pro Arg Glu Glu Ile Arg Tyr His Tyr Ile His Gly Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Ile Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Glu Thr Asn Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Phe Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Phe Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Gly Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp Asn Cys Gly Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ser Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Thr Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Pro Leu Asn Cys Ser Ala Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Arg Val Cys Pro Gly His Asn Phe Cys Ala Glu Ala Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asn Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Asp Cys Gly Ser Gly Gln His Asn Cys Asp Lys Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Gln Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Lys Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ala Glu Gly Phe Val Glu Cys His Asn Tyr Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys His Asn Gly Gln Val Trp Ile Leu Arg Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Asn Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Ser Gly Gln Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Ala Asp Cys Trp Pro Leu Ala Cys Pro Ser Leu Gly Cys Glu Tyr Thr Ala Met Phe Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Gly Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Phe Gly Val Ser Arg Leu Ser Gly Ala Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Arg Val Cys Cys Ser Val Asp Leu Glu Cys Ile Glu Asn Asn Canis lupis familiaris NELL1 amino acid sequence (SEQ ID NO: 15) Met Thr Ser Thr Ser Phe Leu Leu Trp Leu Gly Cys Val His Asn Thr Lys Phe Pro Phe Pro Leu Val Leu Val Thr Arg Ala Ile Val Val Val Val Val Glu Val Val Gly Val Gly Ser Pro Gly Val Arg Ile Arg SerSer Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys Thr Cys Lys Gly Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Arg Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Asp Ala Cys Pro Pro Leu Asn Cys Ser Glu Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Ser Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Arg Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Arg Ala Phe Cys Gln Glu Gly Cys Arg Tyr Gly Gly Ser Cys Val Ser Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Thr Glu Gly Ile Ile Glu Cys His Asn His Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys Arg Asn Gly Gln Val Trp Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Leu Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Ser Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Asn Gly His Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Val Asp Cys Trp Pro Leu Thr Cys Pro Asn Leu Ser Cys Glu Tyr Thr Ala Ile Leu Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Tyr Gly Ile Gly Asp His Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Ser Lys Asn Cys Gln Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Leu Leu Asn Cys Ser Glu Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Ser Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Asp Cys Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Arg Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Met Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ala Glu Gly Ile Ile Glu Cys His Ser His Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Val Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys Arg Asn Gly Gln Val Trp Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Ser Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Asn Gly Asn Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Val Asp Cys Trp Pro Leu Thr Cys Pro Ser Leu Ser Cys Glu Tyr Thr Thr Ile Leu Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Tyr Gly Leu Ser Arg Leu Ser Gly Ser Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Ser Val Cys Cys Ser Val Asp Leu Glu Cys Leu His Asn Asn Ovis aries NELL1 amino acid sequence (SEQ ID NO: 16)Met Pro Met Asp Val Ile Leu Val Leu Trp Phe Cys Val Cys Thr Ala Arg Thr Val Leu Gly Phe Gly Met Asp Pro Asp Leu Gln Leu Asp Ile Ile Ser Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val Ala Gly Leu His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Gln Arg Glu Ile His Ser Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Pro Arg Glu Glu Ile Arg Tyr His Tyr Ile His Gly Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Ile Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Glu Thr Asn Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Phe Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Phe Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Gly Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp Asn Cys Gly Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ser Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Thr Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Pro Leu Asn Cys Ser Ala Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Arg Val Cys Pro Gly His Asn Phe Cys Ala Glu Ala Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asn Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Asp Cys Gly Ser Gly Gln His Asn Cys Asp Lys Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Gln Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Lys Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ala Glu Gly Phe Val Glu Cys His Asn Tyr Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys His Asn Gly Gln Val Trp Ile Leu Arg Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Asn Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Ser Gly Gln Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Ala Asp Cys Trp Pro Leu Ala Cys Pro Ser Leu Gly Cys Glu Tyr Thr Ala Met Phe Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Gly Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Phe Gly Val Ser Arg Leu Ser Gly Ala Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Arg Val Cys Cys Ser Val Asp Leu Glu Cys Ile Glu Asn Asn Homo sapiens NELL1 fragment amino acid sequence (SEQ ID NO: 17) Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile Val Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Ala Gln Val Ser Gly Met His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Ile Glu Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Ile Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Ile His Asn Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Val Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Asp Thr Asn Leu Pro Pro Gly Ile Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Tyr Ile Thr Gln Cys Pro Asn Leu Asn His Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Ser Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Met Cys Pro Pro Leu Asn Cys Ser Glu Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Arg Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Ser Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Arg Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ser Glu Gly Ile Ile Glu Cys His Asn His Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Equus caballus NELL1 fragment amino acid sequence (SEQ ID NO: 18) Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile Ile Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val Ser Gly Leu His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Glu Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu Asn Ser Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Thr His Lys Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Arg Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Ile Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Thr Pro Glu Thr Asn Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Tyr Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Ser Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Val Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Pro Leu Asn Cys Ser Asp Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Ser Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys Arg Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ser Cys Arg Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Thr Glu Gly Ile Ile Glu Cys His Asn His Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser  Bos taurus NELL1 amino acid sequence (SEQ ID NO: 19) Met Ala Leu Cys Ser Phe Ser Val Val Gly Phe Gly Leu Asp Pro Asp Leu Gln Leu Asp Ile Ile Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val Ser Gly Leu His Asn Thr Ser Lys Ala Phe Leu Phe Gln Asp Ala Glu Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Val His Ser Gly Arg Pro Arg Thr Glu Ala Leu Pro Tyr Arg Leu Ala Asp Gly Gln Trp His Arg Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Ile Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Glu Thr Asn Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Tyr Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Ser Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ala Gly Glu Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Ser Lys Ser Cys Gln Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Leu Leu Asn Cys Ser Glu Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Ser Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Asp Cys Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Arg Gly Met Pro Glu Val Gly Pro Pro Arg Ala Leu Leu Asn Ser Leu Asp Leu Gly Phe Leu Ser Phe Ser Lys Glu Ala Leu Ala Val Gly Met Ile Thr Leu Glu Gly Asn Ile Val Ala Lys Ser Phe Thr Asp Asp Glu Thr Leu Val Glu Arg Gly Arg Glu Lys Val Ile Ala Leu Leu Phe Ser Trp Leu His Lys Glu Lys Leu Ser Leu Glu Asn Leu Arg Asp Ile Tyr Cys Lys Ala Asn Ser Leu Val Gly Leu Asp His Leu Pro Gln Arg 

EXAMPLES

The present invention, thus generally described, will be understood morereadily by reference to the following Examples, which are provided byway of illustration and are not intended to be limiting of the instantinvention. The Examples are not intended to represent that theexperiments below are all experiments performed.

Example 1 Cloning and Expression of Recombinant Horse NELL1 Peptides

Various Nell 1 polypeptides were designed based on the horse (Equuscaballus) reference sequence: XP_001505306.1 GI: 149719523 (Aug. 14,2013). The horse protein sequence has recently been updated in the NCBIdatabase as XP_014597419 (Nov. 20, 2015).

The “full-length” version coding sequence begins at amino acid (aa) #3which is the start of the signaling sequence till the last amino acid(#791) and contains all predicted conserved protein domains. Threevariant NELL1 proteins were designed such that certain specific N- orC-terminal domain(s) was/were missing and the alternative form wasshorter in size than the full-length version (Table 2). These fourdifferent recombinant NELL1 proteins were manufactured using ahigh-throughput wheat germ cell-free translation system (commercialCell-Free System (CFS) In Vitro Wheat Germ System developed by AbnovaCorporation, Taipei, Taiwan). The gene sequence was cloned in a plasmidvector that was transcribed and then translated in vitro in a wheat germextract containing all 20 different amino acids. This protein expressionsystem was pioneered by Yaeta Endo and later developed into a highthroughput format (Madin et al. (2000) Proc. Natl. Acad. Sci. U.S.A.97(2):559-564; Sawasaki et al. (2000) Nucleic Acids Symp Ser 44:9-10;Sawasaki et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99(23):14652-14657;and Endo and Sawasaki (2003) Biotechnol. Adv. 21(8):695-713).Purification of the products was accomplished by binding of the NELL1protein tagged with glutathione S-transferase (GST) to an anti-GSTresin. The products were eluted in 50 mM Tris-HCl, 10 mM reducedglutathione, pH 8.0.

TABLE 2 Description of recombinant horse NELL1 peptides. Concen- NELL1Protein Deleted MW tration Purity Description Domains (kDa)* (μg/μl) (%)NELL1 aa 3-791 None 112.42 0.09 87.22 (full-length) NELL1 aa 252-791Entire N- 85.03 0.05 56.27 terminal thrombospondin domain NELL1 aa 3-612Two C-terminal 92.73 0.13 89.44 von Willebrand factor C (VWC) domainsNELL1 aa 34-612 Initial part of N- 89.32 0.17 65.82 terminalthrombospondin domain and 2 C- terminal VWC domains *includes GST-tag of26 kDa

To determine purity, 0.5 microgram protein was loaded in a lane on a12.5% SDS-PAGE gel and stained with Coomassie Blue. BioSpectrum AC®Imaging System with software-VisionWorksLS V6.8 was employed todetermine the purities by calculating the ratio of the major expectedband for each protein variant relative to other minor bands. The valueof concentration is the index of yield. The higher the concentration,the more yield is obtained. If a polypeptide has <0.1 μg/μl, the proteinis considered difficult to produce and purify.

The full-length horse NELL1 protein (aa 3-791) has both lowerconcentration/yield and purity (0.09 μg/μl; 87.22%), compared to theNELL1 variant with 3-612 amino acid sequence (0.13 μg/μl; 89.44%). Theother NELL1 variants (MW=85.03 kDa and MW=89.32) have very lowconcentration/yield and/or purity and were not deemed optimal fortesting further for biological activity in vivo.

Additional independent preparations of variant and full-length NELL1proteins were produced and utilized to check biological activity usingin vitro (e.g. elution of protein from two scaffolds and wound healingscratch assays) and in vivo experiments (e.g. horse body wound healingstudy). Several preparations of amounts ranging from 20 micrograms-2.5milligrams consistently yielded levels of concentration and puritiessimilar to those obtained in the first experiment—>90% for the NELL1variant #3-612 and 85-87% for the full-length (FIGS. 2A-2D).

EXAMPLE 2 Effects of NELL1 Peptide on an In Vitro Model of Tendon Injuryand Repair

There are several in vitro systems that can be utilized to demonstratethe efficacy of NELL1 in tendon repair and regeneration. Thesestrategies make use of precursor or stem cells that give rise to tendonsand can show that addition of NELL1 protein stimulates proliferation,differentiation, gene expression of key genes in tendon formation and/ormigration of these cells.

The effects of NELL1 in the in vitro model of tendon injury and repairas described in Nemoto et al. (2013) J Equine Sci 24(2):17-24 aredetermined as follows. Fibroblasts are obtained from tendon explantsdissected and minced into pieces from the superficial digital flexortendon of healthy adult horses at the time of slaughter. Treatment with0.1% type I collagenase (37 degrees, 20 minutes) will degrade thecollagen holding cells in the tendon ECM, thereby releasing cells. Cellsare cultured on Dulbecco's Modified Eagle Medium [with 10% fetal bovineserum, 100 U/ml penicillin G, 100 microgram/ml streptomycin] at 5% CO₂,37° C. to sub-confluence. Cell cultures are dispersed with 0.1% trypsinin PBS and sub-cultured close to confluence (˜4 days in 12-well plates),then used for the classic scratch assay. Five parallel 0.1 mm“scratches” are made (1000 microliter blue pipette tip) in each cellculture plate, simulating a “wound gap” or injury in the cell sheet.

NELL1 protein, at varying doses, is added to three cultures per dose (15gaps or wounds per dose measured). Three cultures are designated asnegative controls. Initial doses for testing based on other in vitrostudies with NELL1 are (ng/mL): 10, 31.6, 100, 316, 1000, and 3160.

The gaps are examined and measured every other day until they areclosed. Data is analyzed to determined rates of closure and whichtreatment(s) exhibited the best and fastest healing.

The experiment described above is repeated and instead of quantitativemeasurements of gap closure, samples are cultured and harvested atvarious time points (after 0, 12, 24, 28 and 78 hrs post NELL1treatment) for RNA extraction, cDNA synthesis and gene expressionanalysis. Genes that are biomarkers for tendon proliferation andmigration are assessed. Examples of such genes are: Collagen 1, CollagenIII, Tenascin C, and COMP.

EXAMPLE 3 NELL1 Peptide Promotes Cell Migration and Wound Healing inHuman Fibroblasts In Vitro

The effects of two NELL1 proteins (full-length and variant 3-612aa) oncell migration and wound healing were tested and compared using in vitromodels of primary human fibroblasts: adult dermal fibroblasts, type 1diabetes dermal fibroblasts, and ligament fibroblasts. Three doses ofboth NELL1 proteins (full-length and variant 3-612aa) were tested onwound healings assays for each cell type using 4-6 replicates per dose:100 ng/ml, 200 ng/ml, 300 ng/ml. Human fibroblasts were cultured intypical fibroblast growth media (FGM) supplemented with fetal bovineserum (FBS) and various growth factors. Cells in the logarithmic growingphase were cultured overnight in 96-well plates to make cell monolayers.Wound areas (750-micron wide) were generated in the middle of the cellmonolayers in a consistent manner using an IncuCyte WoundMaker® 96(Essen Bioscience, Michigan, U.S.A.). After treatment, cells wereincubated and observed in an IncuCyte® Live-Cell Analysis System (EssenBioscience, Michigan, U.S.A.). Images of cell migration and woundclosure were captured using phase contrast microscopy each hour over a24-hour period. Wound areas, cell confluence and healing rates (averagevelocity of cells moving into gap) were measured and data was analyzedusing GraphPad Prism 7.0.

Not only did the NELL1 variant protein increase the rate of normal humandermal fibroblast cell migration, but also the migration of type 1diabetes dermal fibroblast cells and in a dose dependent manner comparedto the full-length NELL1 protein and control untreated cells, with thehighest dose tested showing the highest increase in wound closure (FIGS.3A-3C). The mean migration rates were: 5.7, 6.4, 6.8, and 8.1 for mediumcontrol, 100, 200, and 300 ng/ml of NELL1 variant, respectively.Treatment with the NELL1 variant at the highest dose tested (300 ng/ml)also showed increased wound healing in ligament-derived fibroblastscompared to untreated controls (FIG. 3D).

Although full-length NELL1 protein did not promote closure of the woundareas in these initial studies, an alternative dosage of full-lengthNELL1 protein or alternative culture conditions, for example, growth ofthe fibroblasts in serum-free medium, might be necessary in order toexhibit such an effect.

Methods

Cells. The following primary cells were purchased: normal human dermalfibroblasts (ixcells Biotech, Cat. #10HU-014), type 1 diabetes humandermal fibroblasts (ixcells Biotech, Cat. #10HU-014), and normal humanligament fibroblasts (ScienCell Research Lab, Inc., Cat. #2630).

Primary cultures of these human fibroblasts were established in completeFibroblast Growth Medium (FGM; ScienCell Research Laboratories; Cat.#2301) and incubated in a humidified 37° C., 5% CO₂ incubator. The FGMwas supplemented with 10% heat-inactivated fetal bovine serum (FBS),penicillin (100 units/ml), streptomycin (100 μg/ml), and other growthfactors, according to the protocol of the cell suppliers.

Human fibroblast monolayer scratch (wound healing) assay. 96-well plateswere coated with a thin layer of biomatrix according to cell types. Thecell numbers were optimized/titrated per well in the plate for each typeof cells. Cells were seeded in the log growing phase into each well(maximum of 100 μL per well) on 96-well microplates and cells wereincubated overnight in a humidified 37° C., CO₂ incubator to form 100%confluent cell monolayers.

An IncuCyte® WoundMaker device/platform was used to create homogeneous,750-micron wide scratch wounds in the middle of the cell monolayers on96-well microplates, by strictly following the IncuCyte® WoundMakerprotocol. The medium with cell debris was removed immediately afterwounding/scratching by gentle aspiration on the side of each well. Eachwell was carefully replenished with 100 μL per well of fresh pre-warmedmedium and any cell debris removed by gentle aspiration on the side ofeach well. Each well (free of debris) was replenished with 50 μL perwell of fresh pre-warmed medium, or 50 μL per well of fresh medium withNELL1 protein (full-length or NELL1 3-612aa variant) with theappropriate concentration to achieve the required dose in each well.Four dosages were tested in at least 4 replicates per treatment:untreated Control (0), 100 ng/mL, 200 ng/mL and 300 ng/mL for each ofthe NELL1 full-length protein and the NELL1 variant protein. Plates weregently tapped to mix.

The plate with cells was incubated in an IncuCyte® Live-Cell AnalysisSystem. Phase contrast images of cell migration towards the empty spacein the wounded area were captured/recorded (10× magnification, digitallyzoomed) at time of 0 (start), and then every other hour post woundingand treatments. The time courses of migration of cells in an IncuCyte®Live-Cell Analysis System were recorded. The total pixel of cells in thewounded area before and after treatments was measured using theIncuCyte™ Software. The following calculations were made from the data:

Wound area at timepoints (Tn)—the area between wound boundaries overtime

Cell confluence area=Wound area (T0)−Wound area (Tn)

Relative cell confluence in the wound=Cell confluence in the wound/totalwound area*100

Wound healing rate=the average velocity at which the cells collectivelymove into the wound gap; is the absolute value of the slope of curvefitting of cell confluence areas over the times (dA/dt) before 50% cellconfluency.

Data were analyzed and plotted out using GraphPad Prism 7.0 (San Diego,Calif.).

EXAMPLE 4 Effects of NELL1 Peptide on an In Vivo Model of EquineTendon/Ligament Injury

Tendon injury animal models are well established in horses because ofthe great demand for treatments of tendon damage, especially in valuableracehorses and show/dressage horses (Schramme et al. (2010) Vet CompOrthop Traumatol 358-365; Estrada et al. (2014) Vet Comp OrthopTraumatol 358-365; Nixon et al. (2008) Am J Vet Res 69:928-937; Watts etal. (2012) EVJ 44(5):576-586). The most common model of damage to theSuperficial Digital Flexor Tendon (SDFT) is a good model for the humanAchilles tendon, a significant and common injury in human patients.Equine SDFT tendonitis can be created chemically using collagenase orvia surgical means.

The following study is performed on a surgically induced SDFT tendonitisequine model.

Twenty-four healthy adult horses are selected and randomly assigned intofour groups with six horses per group. The dosing of NELL1 isextrapolated from wound healing studies in horses based on the amount ofprotein per area of injury. Group 1 is administered saline solution as acontrol. Group 2 is administered dose 1 (400 micrograms of NELL1). Group3 is administered dose 2 (800 micrograms of NELL1) and Group 4 isadministered dose 3 (1600 micrograms of NELL1).

After general anesthesia, a core lesion of 8 cm in the SDFT of arandomized forelimb is generated with a 3.5 mm synovial resector underultrasound imaging. Operated limbs are bandaged for two weekspost-operation until sutures are removed. Horses are confined to a stallduring this two week period.

NELL1 treatment is administered under sedation via intralesionalinjection guided by ultrasound imaging, once at seven days post-injury.

Rehabilitation is performed on a treadmill after two weeks with thelength of time walking steadily increasing (10 mins/day during weeks3-6, 20 mins/day during weeks 7-10, 30 mins/day during weeks 11-14, 40mins/day during weeks 15-20). During weeks 21-22, rehabilitation will be35 mins/day walking and 5 mins/day trotting. Rehab during weeks 23-24will consist of 30 mins/day walking and 10 mins/day trotting.

Healing is assessed by a variety of techniques: a) regular ultrasoundevaluation at 2-weeks, 4-weeks, 6-weeks, 8-weeks, 12-weeks, 16-weeks,20-weeks and 24-weeks; b) histological examination of tendons at the endof study; and c) gene expression via quantitative RTPCR techniques oftendon-specific genes (collagen types I, III, decorin, cartilageoligomeric protein (COMP) and Tenascin C.

EXAMPLE 5 NELL1 Peptide Promotes Healing of Body Wounds in Horse

In order to test the efficacy of the NELL1 protein in healing softtissue injuries in a large animal model with economic/commercialveterinary significance, purified, recombinant, horse NELL1, full-lengthprotein (NFL) and a variant domain-specific form (NV1) were administeredto body wounds of horses. NFL spans amino acids 3-791 of the horse NELL1protein and contains all the known/predicted domains of the horse NELL1protein, while NV1 contains amino acids 3-612 and does not contain thelast two von willebrand factor domains at the C-terminus of the protein.

Six circular 4-cm diameter wounds (3/side) were created surgically onthe thorax of 8 adult female horses to evaluate wound healing. Two daysafter wounding, during the first dressing change, wounds were treatedwith one of the following: control—volume equal to that of sterilesaline that was used to dilute the stock NELL1 protein; dose 1 (95.2μg/4-cm wound) of NFL or NV1; or dose 2 (190.4 μg/4-cm wound) of NFL orNV1. NELL1 was delivered by directly infusing a biodegradable,commercially available calcium alginate dressing. Wound healing wasassessed over a period of 42 days using a 3-D imaging system (Eykonacamera; Bowling et al. (2008) The Eykona Wound Measurement System:Modernizing Wound Measurement for the 21^(st) Century. 12^(th) MalvernDiabetic Foot Conference, UK8, May 14-16; and Bowling et al. (2009)Diabetic Medicine 26(1):93-96). Researchers administering the treatmentand performing the regular wound healing measurements were blinded tothe treatment and dosage given to a wound.

Statistical analyses indicated significant healing effects for thevariant NELL1 protein that enhanced healing based on the remaining innerunhealed wound area at the end of the study (Day 42 post-treatment). Incontrast, there were no statistically significant differences observedbetween untreated control wounds and those treated with the full-lengthNELL1 protein (NFL). Interestingly, from days 3-14 post-treatment,wounds treated with NFL had consistently lower unhealed areas comparedto controls, but these effects were not increased or sustained until theend of the study.

Six general health parameters were monitored daily throughout the studyto evaluate safety: body temperature, pulse rate, respiratory rates,defecation, appetite and behavior/attitude (irritability, aggressivenessor unusual behavior). Results indicated that both NFL and NV1 NELL1proteins did not elicit adverse effects in the test subjects andtherefore, is a safe product to use for wound healing in horses.

This initial equine study suggested that healing effects might beboosted with a second application of the NELL1 protein within the secondweek. There were strong trends of increased wound healing observed fromdays 3-14 with both NELL1 proteins, but these trends were not sustainedfor the full-length NELL1. Future wide range dose optimization studiesmight provide a statistically significant effect with full-length NELL1and also enhance the observed effects of the variant NELL1 protein.

Methods

Horses. Eight mixed-breed, female adult horses, 8 to 15 years old,weighing 453-589 kgs, free of any clinically detectable medicaldisorder, were housed in individual stalls and were kept under constantconditions (i.e., temperature, feeding, cleaning) throughout the study.They were examined daily for signs of discomfort, lameness, and illness.Horses were randomly assigned numbers (referred to as treatmentidentification numbers) 1 through 8, and based on their number, receivedpre-determined grid pattern of wounds, outlined in FIG. 4, andtreatments, listed in Table 3.

Wounding Protocol and Treatment Groups. All procedures were carried outaccording to approved IACUC protocol (UTK-IACUC No. 2247-0314). Thewound model used was a modification of an established equine model(Schumacher et al. (1992) Am J Vet Res 53(9):1568-1571; Gomez et al.(2004) The Canadian Journal of Veterinary Research 68:49-55; and Morganet al. (2009) J Am Vet Med Assoc 234(9):1-8). On day 0, each horse wassedated with detomidine HCl (0.006-0.012 mg/kg) and butorphanol(0.006-0.012 mg/kg). Hair on the right and left sides of the thorax wasremoved with clippers, and the sides of the thorax were prepared foraseptic surgery. Sites of wounds were desensitized with a localanesthetic (2% mepivacaine HCl) subcutaneously. Three, 4-cm diameter andone or two, 2-cm diameter, circular, full-thickness, cutaneous defectswere created on the dorsal aspect of the right and left sides of thethorax (FIG. 4). The 4-cm diameter wounds were created at least 8 cmapart from each other, and 2-cm diameter biopsy wounds were created atleast 4 cm ventral to the most ventral 4-cm diameter wound(s). Allwounds were created using sterilized, 4-cm and 2-cm diameter, stainlesssteel, custom-made, biopsy punches. 4-cm diameter wounds were allowed toheal over 42 days and were assessed qualitatively and by quantitativemeasurements (FIG. 5). The large wounds were arranged in a triangularpattern, as illustrated in FIG. 4, designed to prevent solutions appliedto the dorsally located wounds from migrating by gravity to the ventralwounds. The smaller wounds generated for biopsy were located directlyventral to the ventral large wounds and were treated identically to thelarge wounds. Biopsy tissues were collected for future molecularanalyses.

Wounds were covered with a sterile, alginate dressing cut to the size ofthe wound. The dressings were held in place with a sterile, non-adhesivedressing (Telfa Pad, Johnson & Johnson, New Brunswick, N.J.), which wasstapled to the skin beneath it. Each side of the thorax was covered witha large, sterile combine to compress the wound and to absorb exudate, ifany, from the wound. The dressings and the combines were held in placewith a custom-made, belly bandage (Boa Abdominal Bandage; Wire 2 WireVet Products, LLC, Lexington, Ky.) positioned over the right and leftsides over the thorax, directly over the wounds. Horses receivedphenylbutazone (2.2 mg/kg, IV) before surgery and for 1 day aftersurgery (2.2 mg/kg, orally, q12 h).

On day 2 after wounding, the alginate dressings were carefully removed.NELL1 protein of the appropriate dose (FIG. 4, Table 3) or sterileisotonic saline solution (control) was loaded into the alginate dressingcut to the size of the wound. The NELL1-soaked dressing or the isotonicsaline solution-soaked dressing was laid directly on the wound andcovered with a fresh, sterile, non-absorbent non-adhesive dressing,which was stapled to the skin beneath it. The dressings were removed 2days later, and thereafter, the wounds were dressed twice weekly with asterile combine pad applied to each side of the thorax and held in placewith the belly bandage. FIG. 4 illustrates the position and designationsof the wounds, and Table 3 indicates the treatment applied to eachwound.

TABLE 3 NELL1 form and dosage for treatment of horse wounds. 2-cmdiameter Horse 4-cm diameter wound for # wound biopsy Treatment Dose 1-4A-right side Control NA B-right side NELL1, full- 1 = 95.2 μg length(NFL) C-right side NELL1, Variant 1 1 = 95.2 μg (NV1) W-right sideControl NA X-right side NFL 1 = 23.7 μg E-left side Control NA F-leftside NFL 2 = 190.4 μg G-left side NV1 2 = 190.4 μg Y-left side NV1 2 =47.4 μg 5-8 A-left side control B-left side NFL 1 = 95.2 μg C-left sideNV1 1 = 95.2 μg W-left side NV1 1 = 23.7 μg E-right side Control NAF-right side NFL 2 = 190.4 μg G-right side NV1 2 = 190.4 μg Y-right sideControl NA Z-right side NFL 2 = 47.4 μg Following the treatment groupsshown in Table 3, the total samples per group were: Control = 16 big(4-cm) wounds NFL Dose 1 = 8 big wounds NV1 Dose 1 = 8 big wounds NFLDose 2 = 8 big wounds NV1 Dose 2 = 8 big wounds

Horse NELL1 protein and variants. Purified recombinant horse NELL1proteins were manufactured as described in Example 1. The efficacy oftwo forms of NELL1 protein in treating equine wounds was tested. Thesetwo forms were the full-length protein (aa3-791), designated as NFL, andone shorter domain-specific variant (aa3-612), designated as NV1. TheNELL1 proteins were stored in buffered solution and shipped frozen indry ice from Abnova Corp. for storage at −80° C. until ready for use.These proteins were used within 4 weeks of delivery.

Dosage and Delivery. Wounds were treated with a control (isotonic salinesolution), NELL1 full-length protein, or the NELL1 variant protein twodays after wounding, during the first dressing change. Control treatmentwas phosphate buffered saline (PBS). Dose 1 (i.e., NELL1 full-lengthprotein) was 95.2 micrograms per 4-cm diameter circular wound, and Dose2 (i.e., NELL1 variant protein) was 190.4 micrograms per 4-cm diameterwound.

NELL1 proteins were thawed on ice, proper concentrations were prepared,and the buffered NELL1 proteins were loaded/pipetted directly into theselected dressing. The NELL1 protein was loaded in a volume of 2400 μLfor application to each of all of the 4-cm diameter wounds, and in avolume of 600 μL for application to each of the 2-cm diameter wounds.These volumes were determined by loading the dressings cut to the sizesof the wounds and by testing various volumes of phosphate-bufferedisotonic saline solution to determine maximum loading volume without thesolution dripping from the dressing. A 100-mm diameter sterile plasticPetri dish was used for each dressing, and all protein-soaked dressingswere prepared under sterile conditions in a tissue culture hood. EachPetri dish was labelled with the horse treatment identification numberand the type and dose of the protein applied.

Evaluation of Wound Healing 3-D Imaging. Wound measurements were takenfrom images obtained from a digital, 3-D wound imaging device thatenables rapid, secure, repeatable collection of wound data (TOMI 3D,Eykona Medical, USA). Images were acquired at the following time points:time of wounding, initial treatment, each dressing change (i.e., twiceweekly), and at the termination of study.

Data Analyses. All images were downloaded onto a computer with imageanalysis software developed by Eykona. The images were then analyzedindividually using a trace-area function in the software. The outer mostmargin of the wound was measured first and the area recorded in mm².These values were recorded over the various time points to determine therate of wound contraction. The inner area, or non-epithelialized area,was then measured in similar fashion by tracing the area outlined by theepithelial margin. These values were recorded over time and used tocalculate the rate of epithelialization.

Because there was some degree of variation in the initial size of thewounds, all measurements were normalized on a percent scale so that theinitial wound measurement reflected 100% of the size of the wound.Changes in the overall area of the wound and of the epithelialized areawere then subsequently expressed as a percentage. Rates of contractionand epithelialization were expressed as mm²/day.

Rate of contraction was calculated by subtracting the area of the woundat one time-point from the determined area of the wound at the previoustime-point and then dividing the difference in area by the number ofdays between those two time-points. An overall rate of contraction wascalculated by subtracting the final area of the wound from the initialarea of the wound and dividing the difference by the total number ofdays between those two time-points. The area of epithelialization foreach 4-cm diameter wound was calculated by subtracting the area of thewound from the area of granulation tissue, and the rate ofepithelialization was then calculated in similar fashion by subtractingthe area of epithelialization at one time-point from the area ofepithelialization at the previous time-point and then dividing thedifference in area by the number of days between those two time-points.

Results and Discussion

Overall Health Effects of NELL1 Protein. All horses were monitored dailythroughout the study period. No adverse effects on the health andbehavior of the horses were observed. The temperature, pulse andrespiratory rates, defecation, appetite, and attitude of the horses weremonitored daily and were within normal limits throughout the 42-daystudy.

Delivery of NELL1 protein via calcium alginate dressing. This study isthe first in which NELL1 protein was administered into soft tissueinjuries in horses using a calcium alginate dressing. The selection ofthis dressing was based upon a previous in vitro study comparing therelease of NELL1 protein from a collagen and a calcium alginatecommercial dressing. The elution profiles suggested that in a calciumalginate carrier, the NELL1 protein is released within 2-3 days. In thisin vivo study, at day 3 post-treatment there was no visual evidence ofthe dressing and no abnormal acute inflammatory reactions were observed.

Effects of NELL1 on Wound Healing (Days 3-42, rates of wound healing,epithelialization and contraction). For all data analyses, at a giventime point, the total area of the wound, the inner area (i.e. the areafilled with granulation tissue) and the epithelialized area (i.e., thedifference between the total area and the inner area) were calculatedfor each 4-cm diameter wound of each horse from images obtained at thetime of each bandage change. Each measurement was represented as achange in mm² relative to the previous measurement.

Data was also normalized to account for variation in wound sizes at day0 and considers that all wounds are at 100% value (all unhealed areas)before treatment.

For statistical analyses, cumulative data from each treatment group wereanalyzed. Initially, standard T-TEST using Microsoft Excel (Windows 7)was used to compare these changes, and the p values were calculated. Atday 42 (last day of observation/end of experimental part), analyses ofnormalized data of inner area (unhealed) indicated strong trends in thewounds treated with the variant NELL1 form (p=0.08 for both Dose 1 andDose 2; p=0.04 for all wounds treated with the variant regardless ofdose). Data shows that horse wounds at Day 42 treated with NELL1 variantshowed faster healing (smaller unhealed inner area) compared to control.The wounds have smaller areas that remain unhealed and thevariation/standard deviation was consistently lower in wounds treatedwith the smaller NELL1 variant compared to controls and those treatedwith the full-length form (FIGS. 5 and 6 and Table 4).

TABLE 4 Healing of 4-cm horse body wounds based on the remainingunhealed inner area at various days post-treatment with a degradablecalcium alginate dressing infused with saline solution (untreatedcontrols), full-length (amino acids #3-791) or variant (amino acids#3-612) horse NELL1 protein. OBSER- VATIONS MEAN STANDARD (N) (mm²)DEVIATION CONTROL (UNTREATED) Day 3 (T3) 16 90.1 5.9 Day 7 (T7) 16 76.710.5 Day 14 (T14) 16 39.2 9.5 Day 42 (T42) 16 9.8 5.4 FULL-LENGTH NELL1:DOSE 1 T3 8 85.8 4.9 T7 8 71.1 9.7 T14 8 32.1 6.2 T42 8 10.3 7.9FULL-LENGTH NELL1: DOSE 2 T3 8 81.7 7.5 T7 8 72.3 10.9 T14 8 41.0 6.5T42 8 11.0 9.9 VARIANT NELL1 (aa #3-612): DOSE 1 T3 8 85.3 8.6 T7 8 72.38.4 T14 8 31.2 14.6 T42 8 5.5 3.9 VARIANT NELL1 (aa #3-612): DOSE 2 T3 886.8 5.7 T7 8 72.2 4.3 T14 8 35.5 4.0 T42 8 6.2 2.8

Wounds treated with full-length NELL1 did not show significantdifferences or trend towards better healing at day 42. Interestingly,wounds treated with full-length NELL1 showed significant differences inhaving a smaller inner unhealed area with Dose 2 at 3 dayspost-treatment (p=0.03)

Because wound healing is not a linear process and previous reportssuggested plotting log-transformed values, data was converted into logvalues and plotted (Gelfand et al. (2002) The Society for InvestigativeDermatology 119:1420-1425). These log values were then used forgraphical representation of the data (FIG. 6). Consistent with theinitial analyses, the graphs for wounds treated with the smaller variantshowed a trend for better healing from the controls and at after day 14,these wounds start to heal faster than the other treatment groups.

In addition to the standard T-test, additional statistical tools andapproaches were used to analyze the data. A mixed-model ANOVA was usedto analyze the data to factor in fixed and random effects and the studydesign where a single horse was given several different treatments (2NELL1 protein forms and 2 doses per form).

When treatments were analyzed individually, there are significantdifferences by the mean rate of healing through time and these timepatterns differ by treatment. This was also observed for the otherparameters such as rate of contraction, rate of epithelialization, andoverall healing rate. The mixed model ANOVA analyses confirmed theearlier results where treatment with NELL1 variant showed faster healing(smaller unhealed inner area) compared to control.

Summary and Conclusions

NELL1 horse proteins did not elicit any adverse effects on the wounds,over-all health and behavior of the horses. Horse NELL1 protein isdeemed to be a safe product in the manner delivered and dosages testedin this study. This is the first known application of the horse NELL1protein into an animal model.

NELL1 protein can be effectively and efficiently delivered into horsebody wounds via a calcium alginate biodegradable dressing. The dressingenables delivery into the soft tissue injury within three days, whereinthe entire dressing was completely absorbed into the wounds (byvisual/macroscopic examination). This study represents the first knowntest in vivo of a calcium alginate dressing to administer NELL1 proteininto soft tissue injuries.

NELL1 variant protein exhibited effects that promoted wound healing asmeasured by the remaining inner unhealed area assessed at day 42post-treatment. The standard deviation for treatment at both doses wasvery low compared to either control or full-length NELL. There was nostatistically significant difference between the two tested doses.

There were no statistically significant effects nor strong trends forcontrol wounds or those treated with full-length NELL1 (except for day 3post-treatment for NFL Dose 2, p=0.03) and the variations were large forboth of these groups compared to the NELL1 variant form.

The mixed model analysis indicated that the pattern of healing over timewas significantly different between the various treatment groups. Thelog graphs of inner unhealed area size for the different groups showedthat the NELL1 variant treated wounds started differentiation orseparation from the other treatments after day 14. These data suggestedthat perhaps a re-application of the NELL1 protein (before the end of 14days) might sustain the effects beyond the initial weeks and yield moredramatic effects than initially observed in this study.

Those skilled in the art will further appreciate that the presentinvention may be embodied in other specific forms without departing fromthe spirit or central attributes thereof. In that the foregoingdescription of the present invention discloses exemplary embodimentsthereof, it is to be understood that other variations are contemplatedas being within the scope of the present invention. Accordingly, thepresent invention is not limited to the particular embodiments that havebeen described in detail herein. Rather, reference should be made to theappended claims as indicative of the scope and content of the invention.

1. A variant NELL1 peptide having at least 75% sequence identity to SEQID NO: 17 or 18, wherein said variant NELL1 peptide has at least one ofthe properties selected from the group consisting of enhanced efficacyin tissue regeneration, promotion of wound healing, easier purification,higher yield, and less aggregate formation, when compared to afull-length NELL1 protein.
 2. A variant NELL1 peptide lacking at leastone of the carboxy-terminal von Willebrand factor, type C (VWC) domainsof a NELL1 protein.
 3. The variant NELL1 peptide of claim 2, whereinsaid variant lacks both carboxy-terminal VWC domains.
 4. The variantNELL1 peptide of claim 2 or 3, wherein said variant lacks thecarboxy-terminal 179 amino acid residues of a naturally-occurring NELL1protein.
 5. The variant NELL1 peptide of claim 4, wherein said variantNELL1 peptide comprises the amino acid sequence set forth in SEQ ID NO:17 or
 18. 6. The variant NELL1 peptide of claim 4, wherein said variantNELL1 peptide consists essentially of the amino acid sequence set forthin SEQ ID NO: 17 or
 18. 7. A nucleic acid encoding a variant NELL1peptide of any one of claims 1-6.
 8. A pharmaceutical compositioncomprising the variant NELL1 peptide of any one of claims 1-6 or thenucleic acid of claim
 7. 9. A kit comprising the variant NELL1 peptideof any one of claims 1-6 or the nucleic acid of claim
 7. 10. A methodfor regenerating injured tissue by administering an effective amount ofthe variant NELL1 peptide of any one of claims 1-6, or the nucleic acidof claim 7, to an injured tissue.
 11. A method for promoting thematuration of a progenitor cell by contacting said cell with aneffective amount of the variant NELL1 peptide of any one of claims 1-6,or the nucleic acid of claim
 7. 12. A method for enhancing migration,proliferation, or both migration and proliferation of fibroblast cells,said method comprising contacting said fibroblast cells with aneffective amount of a NELL1 peptide or a nucleic acid encoding a NELL1peptide.
 13. The method of claim 12, wherein said NELL1 peptidecomprises the variant NELL1 peptide of any one of claims 1-6, or thenucleic acid of claim
 7. 14. The method of claim 12 or 13, wherein saidfibroblast cell is a dermal fibroblast or a ligament fibroblast.
 15. Themethod of any one of claims 12-14, wherein migration of fibroblastscomprises migration towards a wound.
 16. A method for promoting thehealing of a wound by administering an effective amount of the variantNELL1 peptide of any one of claims 1-6, or the nucleic acid of claim 7,to a subject in need thereof.
 17. The method of claim 16, wherein saidwound is an open wound.
 18. The method of claim 16 or 17, wherein saidwound comprises an injury to at least one tissue type selected from thegroup consisting of: skeletal muscle, cartilage, bone, and skin.
 19. Themethod of any one of claims 16-18, wherein said variant NELL1 peptide ornucleic acid encoding the same is administered locally to said wound.20. A method for promoting healing of an injury to a tendon or ligamentin a subject in need thereof, wherein said method comprisesadministering an effective amount of a NELL1 peptide or a nucleic acidencoding a NELL1 peptide to said subject.
 21. The method of claim 20,wherein said NELL1 peptide has the amino acid sequence set forth as SEQID NO:
 2. 22. The method of claim 20, wherein said NELL1 peptide has theamino acid sequence set forth as SEQ ID NO:
 4. 23. The method of claim20, wherein said NELL1 peptide has the amino acid sequence set forth asSEQ ID NO:
 6. 24. The method of claim 20, wherein said NELL1 peptide hasthe amino acid sequence set forth as SEQ ID NO:
 10. 25. The method ofclaim 20, wherein said NELL1 peptide has the amino acid sequence setforth as SEQ ID NO:
 12. 26. The method of claim 20, wherein said NELL1peptide is the variant NELL1 peptide of any one of claims 1-6.
 27. Themethod of claim 20, wherein said NELL1 peptide has the amino acidsequence set forth as SEQ ID NO:
 17. 28. The method of claim 20, whereinsaid NELL1 peptide has the amino acid sequence set forth as SEQ ID NO:18.
 29. The method of any one of claims 20-28, wherein the NELL1 peptideor the nucleic acid encoding a NELL1 peptide is administered locally tosaid injury.
 30. The method of any one of claims 20-29, wherein saidNELL1 peptide or nucleic acid encoding a NELL1 peptide is administeredabout seven days post-injury.
 31. The method of any one of claims 16-30,wherein said promotion of healing reduces scarring.
 32. The method ofany one of claims 16-31, wherein said subject is administered anothertreatment for said injury or wound.
 33. The method of any one of claims16-32, wherein said subject is a mammal.
 34. The method of claim 33,wherein said mammal is a human.
 35. The method of claim 33, wherein saidmammal is a horse.
 36. The method of any one of claims 10-35, whereinsaid nucleic acid is comprised within an expression vector and operablylinked to a promoter.
 37. The method of any one of claims 10-36, whereinsaid NELL1 peptide or nucleic acid encoding a NELL1 peptide isincorporated into a drug eluting device, scaffold, matrix, or sutures.